CA2349815A1 - Human cell surface receptor proteins - Google Patents

Abstract

The invention provides human cell surface receptor proteins (HCSRP) and polynucleotides which identify and encode HCSRP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of HCSRP.

Description

HUMAN CELL SURFACE RECEPTOR PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human cell surface receptor proteins and to the use of these sequences in the diagnosis, treatment. and prevention of cell proliferative disorders. immune system disorders. infections. and neuronal disorders.
BACKGROUND OF THE INVENTION
The term receptor describes proteins that specifically recognize other molecules. The categon~ is broad and includes proteins with a variety of functions. The bulk of receptors are cell .surface proteins which bind extracellular ligands and produce cellular responses in the areas of 1o growth, differentiation. endocytosis, exocvtosis and immune response.
Central to the function of cell surface receptors is the capacity to adhere or bind to other proteins or ligands through special functional domains.
Cell surface receptors are typically integral plasma membrane proteins. These receptors recognize hormones such as catecholamines: peptide hormones; growth and differentiation factors;
15 small peptide factors such as thyrotropin releasing hormone; galanin, somatostatin, and tachykinins;
and circulatory system-borne-signaling molecules. Cell surface receptors on immune system cells recognize antigens. antibodies, and major histocompatibility complex (MHC)-bound peptides. Other cell surface receptors bind ligands to be internalized by the cell. This receptor-mediated endocytosis functions in the uptake of low density lipoproteins (LDL), transferrin.
glucose- or mannose-terminal '_'0 glycoproteins, galactose-terminal glycoproteins, immunoglobulins.
phosphovitellogenins. fibrin, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin (Lodish. H. et al.
( 1995) Molecular Cell Bioloey. Scientific American Books, New York NY, p.
723: and Mikhailenko.
I. et al. ( 1997) J. Biol. Chem. 27:6784-6791 ).
Many growth factor receptors, including receptors for epidermal growth factor.
35 platelet-derived growth factor, fibroblast growth factor, as well as the growth modulator a-thrombin, contain intrinsic protein kinase activities. When growth factor binds to the receptor. it triggers the autophosphorylation of a serine. threonine, or t?~rosine residue on the receptor. These phosphorvlated sites are recognition sites for the binding of other cytoplasmic signaling proteins. These proteins participate in siLnalin~; pathways that eventually link the initial receptor activation at the cell surface 30 to the activation of a specific intracellular target molecule. In the case of tyrosine residue autophosphowlation, these signaling proteins contain a common domain referred to as a src homology 2 (SH2) domain. SH? domains are found in a variety of signaling molecules and oncoQenic proteins such as phosphoiipase C-y. Ras GTPase activating protein. and pp60''s"
(Lowenstein, E.J. et al. ( 1992) Cel I 70:131-4:12 ).
G-protein coupled receptors (GPCRs) G-protein coupled receptors (GPCRs) are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which span the plasma membrane and form a bundle of antiparallel alpha (a) helices. These proteins range in size from under 400 to over 1000 amino acids (Strosberg, A.D. ( 1991 ) Eur. J. Biochem. 196:1-10; Coughlin.
S.R. ( 1994) Curr. Opin.
Cell Biol. 6:191-197). The amino-terminus of the GPCR is extracellular, of variable length and often glycosylated; the carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops of the GPCR alternate with intracellular loops and link the transmembrane domains. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops.
The transmembrane domains account for structural and functional features of the receptor. In most cases. the bundle of alpha helices forms a binding pocket. In addition, the extracellular N-terminal segment or one or more of the three extracellular loops may also participate in figand binding. Ligand I S binding activates the receptor by inducing a conformational change in intracellular portions of the receptor. The activated receptor, in turn, interacts with an intracellular heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositoi triphosphate or ion channel proteins (Baldwin, J.M. (1994) Curr.
Opin. Cell Biol. 6:180-190).
One group of GPCRs are the rhodopsin-like GPCRs that transmit extracellular signals of diverse natures including hormones, neurotransmitters and light. Rhodopsin is a photosensitive GPCR in the vertebrate eye. Rhodopsin, which defines a conserved subfamily of GPCRs found in animal retinas, is about 350 amino acids in length. In vertebrates, rhodopsin molecules are embedded in membranous stacks found in photoreceptor (rod) cells. Each rhodopsin molecule responds to a photon of light by triggering a decrease in cGMP levels which leads to the closure of plasma membrane sodium channels. In this manner, a visual signal is converted to a neural impulse. Other rhodopsin-like GPCRs are directly involved in responding to neurotransmitters.
These GPCRs include the receptors for adrenaline (adrenergic receptors), acetylcholine (muscarinic receptors}, adenosine, galanin. and glutamate (N-methyl-D-aspartate/NMDA receptors).
(Reviewed in Watson, S. and Arkinstall. S. ( 1994) The G-Protein Linked Receptor Facts Book, Academic Press, San Diego, CA, pp. 7-9. 19-22, 32-35, 130-131, 214-216, 221-222: Habert-Ortoii. E. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:9780-9783.) The somatostatin receptor type d is another example of a rhodopsin-like GPCR.
It is one of several high affinity receptors for somatostatin. a tetrapeptide that inhibits the secretion of growth hormone from the anterior pituitan~. Expression of particular sornatostatin receptors has been linked to the efficacy of drug therapy in specific endocrine tumors (Kubota. A. et al. ( 1994) J. Clin. Invest.
93:1321-1325).
Another rhodopsin-like GPCR is the prostanoid EP1 receptor that recognizes prostanoids such as prostaglandin to mediate a variety of physiological functions including cardiovascular and immune responses. EP1 receptors have a role in the contraction and relaxation of smooth muscle and can activate the phosphoinositide pathway ( Watson, supra, pp. 239-251 ). The prostanoid DP receptor is another rhodopsin-like GPCR that is specific for prostaglandin D2 (PGD2).
Expression of the DP
receptor has been localized to the mammalian brain and eye tissues and upon activation facilitates elevation of intracellular CAMP and Ca2+ mobilization but does not generate inositol 1,4,5-triphosphate (Bole, Y. et al. (1995) J. Biol. Chem. 270:18910-18916;
Gerashchenko. D. et al. (1998) J. Neurochem. 71:937-945).
Still another rhodopsin-like GPCR is the endothelin receptor that plays a role in cardiovascular system regulation through endothelins. Endothelins are potent vasoconstrictors that can stimulate cardiac and smooth muscle contraction as well as stimulate secretion in tissues such as kidney, liver and adrenals. Endothelin receptors may have a role in the brain, where they are also found, and there is evidence that endothelins may be associated with pathophysiological conditions such as stress (Watson, supra, pp. 111-116).
The secretin receptor is an example of a unique GPCR that responds to secretin, a peptide hormone that stimulates the secretion of enzymes and ions in the pancreas and small intestine (Watson, supra, pp. 278-283). Secretin receptors are about 450 amino acids in length and are found in the plasma membrane of gastrointestinal cells. Binding of secretin to its receptor stimulates the production of cAMP. An unusual member of the secretin receptor family has been identified from a neuroectodermal cDNA library (Baud, V. et al. ( 1995) Genomics 26:334-344).
This receptor. EMR t (EGF-like, mucin-like hormone receptor), is 886 amino acids in length and contains six epidermal growth factor (EGF)-like modules at the N-terminus followed by a serine/threonine rich domain. The latter feature is characteristic of mucin-like integral membrane adhesive proteins.
Other GPCRs have been identified which play a role in the immune response. For example, a new subfamily of GPCRs has been identified from a human monocyte (HM) cDNA
library (Nomura, H. et al. (1993) Int. Immunol. 5:1239-1249). Most of these GPCRs likely bind to cvtokines and other leukocvtic signaling molecules. One of these GPCRs, HM74, is particularly unusual in that its N-terminus does not contain N-glycosylation sites.
The thrombin receptors (TRs) have GPCR activity and are activated by the ligand a-thrombin.
Through TR-mediated signal transduction pathways, a-thrombin induces production of IL-8 and IL-6 in cultured monocytes and endothelial cells (Johnson, K. et al. ( 1998) J.
Immunol. 160:5130-5130.
Conversely, a-thrombin inhibits the action of IL-6. leukemia inhibitory factor. and ciliarv neurotrophic factor in Chinese hamster tuns: fibroblasts (Bhat. G.J. et al. ( 1998) Arch. Biochem.
Biophys. 350:307-311). In addition, when a-thrombin binds to the TR it proteolyically cleaves 40 amino acids from the N-terminus of the receptor. The cleaved peptide is termed the thrombin receptor agonist peptide and acts as a tethered ligand for the TR to increase the potency of the thrombin-derived signal (Hou, L. Et al. ( 1998) J. Periodontal Res. 33:?OS-211;
Johnson, et al. supra).
GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Parma, J. et al. ( 1993, Nature 365:649-651 ) report that somatic activating mutations in the thyrotropin receptor cause hyperfunctioning thyroid adenomas and suggest that certain GPCRs susceptible to constitutive activation may behave as protooncogenes.
Cvtokine Receptors Cytokines comprise a family of signaling molecules that modulate the immune system and the IS inflammatory response. Cytokines are usually secreted by leukocytes, or white blood cells, in response to injury or infection. However, other tissues are capable of secreting cytokines in response to disease or other physiologic perturbations. Cytokines function as growth and differentiation factors that act primarily on cells of the immune system such as lymphocytes, monocytes, and granulocytes.
Like other signaling molecules, cytokines bind to specific plasma membrane receptors and trigger intracellular signal transduction pathways which regulate gene expression, cell proliferation, and cell differentiation.
Erythropoietin (EPO) is an unusual cytokine that is produced not by leukocytes, but instead by the kidney or liver. EPO stimulates erythroid precursors to differentiate into red blood cells. EPO
also stimulates the production of platelets. The EPO receptor is a single-pass transmembrane protein of about 500 amino acids, the intracellular domain of which associates with JAK2 kinase. Activated EPO receptor stimulates the phosphorylation activity of JAK2 which triggers gene transcription and mitogenesis. (Reviewed in Callard, R. and Gearing, A. ( 1994) The Cvtokine Facts Book, Academic Press, San Diego, CA, pp.l 14-118.) immunoalobulin Domain Receptors Immune system and related cell surface receptor proteins have hallmark functional domains (for example the Immunoglobulin (Ig) domain) with similar functionality in a wide array of receptor types. The human immune system is responsible for combating infectious agents contracted from the environment. One critical component of the immune system are highly specialized molecules called immunoglobulins (lg) or antibodies that can recognize and bind to foreign antigens, thereby .t facilitating their elimination. Characteristic features of immunoglobulins include their structural motifs that include regions for membrane attachment. antigen recognition (variable (V) regions), and polymerization. Polymerized immunoglobulins such as glandularly secreted IgA
and IgM must undergo transcellular transport. a process mediated by the poly-immunoglobulin (poly-Ig) receptor.
The poly-Ig receptor is itself a member of the immunoglobulin superfamily having homology to the variable (V) region of immunogiobulins (Hood. L. et al. ( 1985) Cell 40:225-229). Like all immunoglobulin superfamiiy members, poly-Ig receptor is involved in adhesion or binding to other proteins through the conserved immunoglobulin-like domain. This Ig domain is comprised of antiparallel ~3 sheets joined by a disulfide bond in an arrangement called the Ig fold. Members of the Ig superfamily include T-cell receptors, MHC proteins, CD4, CDB, and CD 28 cell surface proteins, and antibodies.
Immunogiobulins, or antibodies. are the central components of the humoral immune response.
IgG, the most common class of immunoglobulin in the circulation. can be described in terms of two main functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the IgG, while effector functions are mediated by the Fc (crystallizable fragment) region.
Binding of IgG to an antigen, such as a bacterium, triggers the destruction of the antigen by phagocytic white blood cells, such as macrophages and neutrophils. These cells express cell surface receptors that specifically bind to the IgG Fc region and allow the phagocytic cells to engulf, ingest, and degrade the IgG-bound antigen. The IgG Fc receptors expressed by phagocytic cells are single-pass transmembrane giycoproteins of about 400 amino acids (Sears. D. W. et al.
( 1990) J. lmmunol.
144:371-378). The extracelluiar portion of the IgG Fc receptor typically contains two or three Ig domains.
T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate other immune cells. Although T cells collectively recognize a wide range of different antigens, a clonal line of T cells can only recognize a single antigen. Moreover, the antigen must be presented to the T cell receptor (TCR) as a peptide complexed with a major histocompatibility molecule (MHC) on the surface of an antigen-presenting cell. The TCR on most T cells consists of two polypeptide subunits, a and p, which are immunoglobulin-like integral membrane glycoproteins of similar molecular weight. The TCRa and TCR~i subunits have an extracellular domain containing both variable and constant regions. a transmembrane domain that traverses the membrane once, and a short intracellular domain (Saito, H. et al. ( 1984) Nature 309:77-762). The genes for the TCR
subunits are constructed through somatic rearrangement of different gene segments. Interaction of antigen in the proper MHC
context with the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components of the immune system ( Weiss. A. ( 1991 ) Annu. Rev. Genet. 25: 487-510). Rearrangements in TCR genes and alterations in TCR expression have been noted in lymphomas, leukemias. autoimmune disorders, and immunodeficiency disorders (Aisenberg, A.C. et al. ( 1985) N. Engl. J. Med. 313:529-X33: Weiss, supra; and Olive, sulara).
Immunizations with peptides derived from TCRs are effective treatment for some human T-cell-mediated autoimmune disease and in animal models of such illnesses, in particular. rheumatoid arthritis (Bridges, S.L. and Moreland. L.W'. (1998) Rheum. Dis. Clin. North Am. 24:641-650).
The discovery of new human cell surface receptor proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention. and treatment of cell proliferative disorders, immune system disorders, infections, and neuronal disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human cell surface receptor IS proteins, referred to collectively as "HCSRP" and individually as "HCSRP-l," "HCSRP-2,"
"HCSRP-3," ''HCSRP-4," "HCSRP-5," "HCSRP-6," "HCSRP-7," "HCSRP-8," "HCSRP-9,"
"HCSRP-10," ''HCSRP-1 1," "HCSRP-12," and "HCSRP-13." In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-13 and fragments thereof. The invention also includes a polypeptide comprising an amino acid sequence that differs by one or more conservative amino acid substitutions from an amino acid sequence selected from the group consisting of SEQ ID NO:I-13.
The invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID
NO:1-l3 and fragments thereof. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:I-13 and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-13 and fragments thereof.
Additionally, the invention provides an isolated and purified poiynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the poiypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-13 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementan- to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: I-I; and fragments thereof.
The invention also provides a method for detecting a polynucieotide in a sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the pofynucleotide sequence to at least one of the polynucleotides of the sample.
thereby forming a hybridization complex: and (b) detecting the hybridization complex. wherein the presence of the hybridization complex correlates with the presence of a polynucieotide in the sample. In one aspect.
the method further comprises amplifying the polynucleotide prior to hybridization.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:14-26 and fragments t0 thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID N0:14-26 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a po(ynucleotide sequence selected from the group consisting of SEQ ID N0:14-26 and fragments thereof.
The invention further provides an expression vector containing at least a fragmem of the polynucleotide encoding the poiypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: t-13. In another aspect, the expression vector is contained within a host cell.
?0 The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing a polynucleotide of the invention under conditions suitable for the expression of the polypeptide: and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: I-13 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO: I-13 and fragments thereof. The invention also provides a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of HCSRP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID
NO:I-13 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.

WO 00/2$032 PCT/US99/26742 The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of HCSRP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: I-13 and t~aements thereof.
BRIEF DESCRIPTION OF THE TABLES
Table i shows polypeptide and nucleotide sequence identification numbers (SEQ
1D NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding HCSRP.
Table 2 shows features of each polypeptide sequence. including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of HCSRP.
Table 3 shows selected fragments of each nucleic acid sequence: the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis;diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding HCSRP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze HCSRP, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins. nucleotide sequences. and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described. as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims. the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates othenvise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined othenvise. all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines. materials. and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols. reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

''HCSRP" refers to the amino acid sequences of substantially purified HCSRP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine. equine. and human, and from any source. whether natural, synthetic. semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of HCSRP. Agonists may include proteins, nucleic acids. carbohydrates, small molecules, or anv other compound or composition which modulates the activity of HCSRP either by directly interacting with HCSRP or by acting on components of the biological pathway in which HCSRP
participates.
An ''allelic variant" is an alternative form of the gene encoding HCSRP.
Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding HCSRP include those sequences with deletions, insertions. or substitutions of different nucleotides. resulting in a polypeptide the same as HCSRP or a polypeptide with at least one functional characteristic of HCSRP. included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HCSRP. and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HCSRP. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HCSRP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as lone as the biological or immunological activity of HCSRP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid.
and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine: and serine and threonine.

Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valise: glycine and alanine: and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide.
peptide, polypeptide. or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of HCSRP. Antagonists may include proteins such as antibodies. nucleic acids, carbohydrates. small molecules, or any other compound or composition which modulates the activity of HCSRP either by directly interacting with HCSRP or by acting on components of the biological pathway in which HCSRP participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')_~ and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind HCSRP polypeptides can be prepared using intact pofypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers chat are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term ''antigenic determinant" refers to that region of a molecule (i.e..
an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal. numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid sequence which is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the Id complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation "neeative"
or "minus" can refer to the antisense strand. and the designation "positive" or "plus" can refer to the sense strand.
The term "biologically active" refers to a protein havin;; structural.
reeulatorv~, or biochemical functions of a naturally occurring molecule. Likewise, "immunoloLically active" refers to the capability of the natural, recombinant, or synthetic HCSRP. or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The terms "complementary" and ''complementarily" refer to the natural binding of polynucleotides by base pairing. For example, the sequence ''S' A-G-T 3"' bonds to the complementary sequence "3' T-C-A 5'." Complementarily between two single-stranded molecules may be "partial," such that only some of the nucleic acids bind. or it may be ''complete," such that total complementarily exists bet<veen the single stranded molecules. The degree of complementarily between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which IS depend upon binding between nucleic acid strands, and in the design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding HCSRP or fragments of HCSRP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI). detergents (e.g.. sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of one or more Incyte Clones and. in some cases, one or more public domain ESTs.
using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG. Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
''Conservative amino acid substitutions" are those substitutions that. when made, least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
Original Residue Conservative Substitution Ala Gly. Ser Arg His. Lys Asn Asp. Gltt. His Asp Asn, Glu Cys Ala, Ser Gln Asn. Glu, His Glu Asp, Gln. His Gly Ala His Asn, Arg, Gln. Glu lle Leu, Val Leu Ile, Val Lys Arg, Gln, Glu I S Met Leu, l le Phe His, Met. Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution. and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term °derivative" refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegyiation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "fragment" is a unique portion of HCSRP or the polynucleotide encoding HCSRP
which is identical in sequence to but shorter in length than the parent sequence. A
fras~ment may comprise up to the entire length of the defined sequence. minus one nucleotide/amino acid residue. For example, a fragment may comprise from ~ to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer. antigen. therapeutic molecule, or for other purposes.
may be at least ~. 10, I5, 20, 25. 30, .10. 50. 60, 75, 100. 1 S0. 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example.
a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the tirst 250 or X00 amino acids (or first 2~% or ~0% of a polypeptide) as shown in a certain defined sequence. Clearly these lengths are exemplary. and any length that is supported by the specification, including the Sequence Listing, tables. and figures. may be encompassed by the present embodiments.
A fragment of SEQ ID NO:1 '1-26 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: Id-26, for example, as distinct from any other sequence in the same genome. A fragment of SEQ ID N0:14-26 is useful. for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID
N0:14-26 from related polynucleotide sequences. The precise length of a fragment of SEQ ID N0:14-26 and the region of SEQ ID N0:14-26 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO: I-13 is encoded by a fragment of SEQ ID N0:14-26. A
fragment of SEQ ID NO:1-13 comprises a region of unique amino acid sequence that specifically identifies IS SEQ ID NO:1-13. For example, a fragment of SEQ ID NO:I-13 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-13.
The precise length of a fragment of SEQ ID NO:1-13 and the region of SEQ ID NO:1-13 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
The term "similarity'' refers to a degree of complementarity. There may be partial similarity or complete similarity. The word "identity" may substitute for the word "similarity." A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as "substantially similar." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e.. a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.

WO 00/28032 PCT/US99/2b742 The phrases "percent identity" and "% identiy." as applied to polynucleotide sequences. refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert. in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences. and therefore achieve a more meaningful comparison of the tvo sequences.
Percent identity behveen polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR. Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp ( 1989) CABIOS 5:151-153 and in Higgins. D.G. et al. ( 1992) CABIOS
8:189-191. For pairwise alignments of poiynucleotide sequences, the default parameters are set as follows: Ktuple=2. gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequence pairs.
IS Altemativeiy, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.eov/BLAST/. The BLAST software suite includes various sequence analysis programs including ''blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http:/hvwv.ncbi.nim.nih.;ov/gorf/612.html. The "BLAST 2 Sequences" too! can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the ''BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such default parameters may be, for example:
Matrir: BLOSUM62 Reward jor matclr 1 Penaltvjorntismatchw-Z
Open Gup: .5 and E.rtension Gap: 1 penalties Gap x drop-oJf .i0 Expect: 10 Word Si_ e: I 1 ld Filter: on Percent identity may be measured over the length of an entire defined sequence. for example.
as defined by a particular SEQ ID number, or may be measured over a shorter lensth, for example, over the length of a fragment taken from a larger. defined sequence. for instance. a fragment of at (east 20, at least 30, at least 40, at least ~0, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only. and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures. or Sequence Listing.
may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity." as applied to polypeptide sequences. refer to the percentage of residue matches between at least two polypeptide sequences aligned using a IS standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For painvise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=~, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments. the percent identity is reported by CLUSTAL V as the ''percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the ''BLAST 2 Sequences"
tool Version 2Ø9 (May-07-1999) with blastp set at default parameters. Such default parameters may be' for example:
Matrir: BLOSUM62 Open Gup: Il and E.rtension Gap: I penalties Gap x drop-ojj .i 0 Expect: 10 Word Si=e: 3 Filter.' on IS

Percent identity may be measured over the length of an entire defined polypeptide sequence.
for example, as defined by a particular SEQ ID number. or may be measured over a shorter length, for example. over the Ien~,th of a fragment taken from a larger, defined polypeptide sequence, for instance. a fragment of at least 15, at least 20. at least 30, at least d0. at least ~0, at least 70 or at least 1 SO contiguous residues. Such lengths are exemplary only. and it is understood that any fragment length supported by the sequences shown herein. in the tables, fi;ures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain ail of the elements required for stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under perm issive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 ~tg/mi denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Generally. such wash temperatures are selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook et al., 1989, Molecular Cloning A Laboratory Manual, 3"" ed.. vol. I-3, Cold Sprin~~ Harbor Press. Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS. for 1 hour.
Alternatively, temperatures of about 6~°C, 60°C. S~°C. or d2°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC. with SDS being present at about 0.1 °,6. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance.
denatured salmon sperm DNA at about 100-200 pg/ml. Organic solvent. such as formamide at a concentration of about 35-50% v/v. may also be used under particular circumstances. such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity bet,veen the nucleotides. Such similaric is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between t'vo nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.~., C°t or R~,t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid t5 support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease. etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on a substrate.
The terms ''element" and "array element" in a microarray context, refer to hybridizable 35 polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of HCSRP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics. or any other biological, functional, or immunological properties of HCSRP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide.
polynucleotide. or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand. to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operable linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance. a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the codinc sequence. Generally, operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions. in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which ~ comprises an oligonucleotide of at least about ~ nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Probe" refers to nucleic acid sequences encoding HCSRP, their complements, or fragments thereof. which are used to detect identical. allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target IS DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least I S contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed. such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 1 SO consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures. and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel et al., l 987, Current Protocols in Molecular Bioloey, Greene Publ. Assoc. & Wiley-Intersciences, New York NY: lnnis et al., 1990, PCR Protocols. A
Guide to Methods and Applications, Academic Press, San Diego CA. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991. Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 1.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to x,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example. the PrimOU primer selection program (available to the public from the Genome Center at Universin~ of Texas South West Medical Center. Dallas TX) is capable of choosing specific primers from me~abase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT
Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library." in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence. this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for IS example, as PCR or sequencing primers. microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oiigonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed. inducing a protective immunological response in the mammal.
The term "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding HCSRP, or fragments thereof, or HCSRP itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle. or membrane isolated from a cell;
a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue: a tissue print:
etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist. an antibody, an antagonist. a small molecule. or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A." the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides. respectively.
''Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips. slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, I S microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
"Transformation" describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection.
electroporation, heat shock, lipofection, and particle bombardment. The term "transformed" cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%. at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined Length. A variant may be described as. for example, an "allelic" (as defined above), "splice." "species." or "polymorphic"
variant. A splice variant may have significant identity to a reference molecule. but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide poiymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using biastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-! 999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%. at least 60%, at least 70%, at least 80%. at least 90%, at least 9~%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.
IS

The invention is based on the discovery of new human cell surface receptor proteins (HCSRP), the polynucleotides encoding HCSRP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative disorders, immune system disorders. infections, and neuronal disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding HCSRP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding etch HCSRP were identified, and column 4 shows the cDNA
libraries from which these clones were isolated. Cotumn ~ shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA Libraries. The Incyte clones in column 5 were used to assemble the consensus nucleotide sequence of each HCSRP and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of the invention:
column I references the SEQ ID NO: column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites: column 4 shows potential glycosylation sites; column ~ shows the amino acid residues comprising signature sequences and motifs: column 6 shows homologous sequences as identified by BLAST analysis; and column 7 shows analytical methods used to characterize each polypeptide through sequence homology and protein motifs. In particular. the amino acid sequence of SEQ 1D NO: l from about amino acid residue 30 to about 81 is distinct from the tethered ligand thrombin receptor agonist peptide of the N-terminus of the human thrombin receptor and the amino acid sequence of SEQ ID 1v'0:2 from about amino acid residue I I S
to about 140 is distinct from the C-terminus joining and constant regions of the human TCRa subunit.
The columns of Table 3 show the tissue-specificity and diseases. disorders. or conditions associated with nucleotide sequences encoding HCSRP. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1. These fragments are useful, for example. in hybridization or amplification technologies to identify SEQ ID
N0:14-26 and to distinguish between SEQ ID N0:14-26 and related polynucleotide sequences. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 lists tissue categories which express HCSRP as a fraction of total tissues expressing HCSRP.
Column 4 lists diseases, disorders, or conditions associated with those tissues expressing HCSRP as a fraction of total tissues expressing HCSRP. Column ~ lists the vectors used to subclone each cDNA
library. Of particular note is the expression of HCSRP in cancer, autoimmune and inflammatory IS response, and in lung, thymus, bladder. seminal vesicle, and penile tissues, and in rheumatoid arthritis.
In addition, SEQ ID NO: 14 is expressed primarily in tumor-associated epithelial tissues and SEQ ID
NO: 15 is expressed primarily in growth- and tumor-associated epithelial tissues and in immune response tissues.
The columns of Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding HCSRP were isolated. Column 1 references the nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
The invention also encompasses HCSRP variants. A preferred HCSRP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the HCSRP amino acid sequence, and which contains at least one functional or structural characteristic of HCSRP.
The invention also encompasses polynucleotides which encode HCSRP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:14-26, which encodes HCSRP.
The invention also encompasses a variant of a polynucleotide sequence encoding HCSRP. In particular. such a variant polynucleotide sequence will have at least about 80%. or alternatively at least about 90%, or even at least about 9~% polynucleotide sequence identity to the polynucleotide sequence encoding HCSRP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ 1D

N0:14-'_'6 which has at least about 80%. or alternatively at least about 90%, or even at least about 9S% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: l4-26. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HCSRP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code. a multitude of polynucleotide sequences encoding fiCSRP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring HCSRP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode HCSRP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring HCSRP
under appropriately I S selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HCSRP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding HCSRP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode HCSRP
and HCSRP derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HCSRP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular. to those shown in SEQ ID
N0:14-26 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger ( 1987) Methods Enzymol. 152:399-407; Kimmel, A.R. ( 1987) Methods Enzymol.
1 S2:S07-S 1 l .) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of ?3 the embodiments of the invention. The methods may employ such enzymes as the Klenow frasment of DNA polymerase 1. SEQUENASE (US Biochemical. Cleveland OH). Taq polymerase (Perkin-Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech. Piscataway NJ). or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE
~ amplification system (Life Technologies. Gaithersburg MD). Preferably.
sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABf CATALYST 800 thermal cycler (Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. ( 1997) Short Protocols in Molecular Biolo >v, John Wiley & Sons, New York NY, unit 7.7:
Meyers, R.A. (1995}
Molecular Biology and Biotechnolo,y, Wiley VCH. New York NY, pp. 856-853.) The nucleic acid sequences encoding HCSRP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. ( 1988) Nucleic Acids Res. 16:8186.) A third method. capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom. M. et al.
(1991 ) PCR Methods Applic. 1:11 1-t 19.) fn this method, multiple restriction enzyme digestions and ?5 ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. ( 1991 ) Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers. and PROMOTERFINDER libraries (Clontech. Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods. primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences. Plymouth MN) or another appropriate program. to be about 22 to 30 nucleotides in length. to have a GC content of about ~0% or more. and to anneal to the template at temperatures of about 68°C to 72°C.

When screening for full-length cDNAs. it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition. random-primed libraries, wi~ich often include sequences containing the ~' regions of genes. are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into ~' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular. capillary sequencing may employ flowable polymers for electrophoretic separation. four different nucleotide-specific. laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate sofrivare (e.g.. GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer). and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
Capillary electrophoresis is especially preferable for sequencing small DNA
fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode HCSRP may be cloned in recombinant DNA molecules that direct expression of HCSRP, or fragments or functional equivalents thereof. in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express HCSRP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HCSRP-encoding sequences for a variety of purposes including, but not limited to. modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding HCSRP may be synthesized. in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. ( 1980) Nucleic Acids Symp. Ser. 7:215-223: and Horn, T. et al. ( 1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, HCSRP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J.Y. et al. ( i995) Science 269:202-20~.) Automated synthesis may be achieved using the ABI 43 tA peptide synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of HCSRP, or any part thereof. may be altered during direct synthesis and/or combined with sequences from other proteins. or any pan thereot: to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier ( 1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing.
(See, e.g., Creighton. T. ( 1984) Proteins. Structures and Molecular Properties, WH Freeman, New York NY.) In order to express a biologically active HCSRP. the nucleotide sequences encoding HCSRP
or derivatives thereof may be inserted into an appropriate expression vector.
i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and S' and 3' untranslated regions in the vector and in polynucleotide sequences encoding HCSRP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HCSRP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding HCSRP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence. or a fragment thereof, is inserted. exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used.
(See, e.g.. Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding HCSRP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook. J. et al. ( 1989) Molecular Clonine A
Laboratory Manual. Cold Spring Harbor Press, Plainview NY, ch. 4, 8. and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo v, John Wiley & Sons, New York NY. ch. 9, 13, and 16.) 0 A variety of expression vector/host systems may be utilized to contain and express sequences encoding HCSRP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid. or cosmid DNA expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g.. baculovirus);
plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus. CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.~;.. Ti or pBR3?2 plasmids): or animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of clonine and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding HCSRP. For example. routine cloning, subcloning, and propagation of polynucleotide sequences encoding HCSRP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding HCSRP into the vectors multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g.. Van Heeke, G. and S.M.
Schuster ( 1989) J, Biol.
Chem. 264:5503-5509.) When large quantities of HCSRP are needed. e.g. for the production of antibodies, vectors which direct high level expression of HCSRP may be used.
For example, vectors containing the strong, inducible T~ or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HCSRP. A number of vectors containing constitutive or inducible promoters, such as alpha factor. alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichiapastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, supra; Bitter, G.A. et al. ( I 987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. ( 1994) BiolTechnology 12:181-184.) Plant systems may also be used for expression of HCSRP. Transcription of sequences encoding HCSRP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
( 1987) EMBO J.
6:307-3 I 1 ). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. ( 1984) EMBO J. 3:1671-I
680; Broglie, R. et al.
( 1984) Science 224:838-843; and Winter, J. et al. ( 1991 ) Results Probl.
Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technoio v (1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells. a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding HCSRP
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E 1 or E3 region of the viral genome may be used to obtain infective virus which expresses HCSRP in host cells. (See, e.'~., Logan. J.
and T. Shenk ( 1984) Proc.
Natl. Acad. Sci. USA 81:36~~-369.) In addition. transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer. may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes.
polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat. Genet.
15:345-355.) For long term production of recombinant proteins in mammalian systems. stable expression of HCSRP in cell lines is preferred. For example, sequences encoding HCSRP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media IS before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See. e.g., Wigler, M. et al. ( 1977) Cell t 1:223-232: Lowy, 1. et al. ( 1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhJr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and a!s and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. ( 1980} Proc. Natl. Acad. Sci. USA 77:3567-3570: Colbere-Garapin, F. et al. ( 1981 ) J. Mol. Biol. 150: I-14.) Additional selectable genes have been described, e.g., trpB and hisD. which alter cellular requirements for metabolites. (See, e.g.. Hartman, S.C. and R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g.. anthocyanins, green fluorescent proteins (GFP; Clontech), f3 glucuronidase and its substrate f3-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g.. Rhodes. C.A. ( 1995) Methods Moi. Biol. »:1' I-131.) Although the presence/absence of marker gene expression suggests that the Gene of interest is also present. the presence and expression of the gene may need to be contirmed. For example, if the sequence encoding HCSRP is inserted within a marker gene sequence. transformed cells containing sequences encoding HCSRP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding HCSRP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding HCSRP
and that express HCSRP rnay be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to. DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane. solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of HCSRP
using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and IS fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HCSRP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. ( i 990) Seroloeical Methods a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV: Coligan, J.E. et al. (1997) Current Protocols in Immunolo y, Greene Pub. Associates and ~Viley-Interscience, New York NY: and Pound, J.D. ( 1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HCSRP
include oligolabeling, nick translation, end-labeling. or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding HCSRP. or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerise such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides. enzymes, fluorescent, chemiluminescent, or chromogenic agents. as well as substrates, cofactors. inhibitors. magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding HCSRP may be cultured under conditions suitable for the expression and recovery of the protein tcom cell culture. The protein produced by a transformed cell may be secreted or retained intracelluiarly depending on the sequence and/or the vector used. As will be understood by those of skill in the art.
expression vectors containing polynucleotides which encode HCSRP may be designed to contain signal sequences which direct secretion of HCSRP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression ofthe inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding. and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293. and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified. or recombinant nucleic acid sequences encoding HCSRP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric HCSRP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HCSRP
activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP). thioredoxin (Trx). calmodulin binding peptide (CBP), 6-His, FLAG, c-mvc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized giutathione, maltose, phenylarsine oxide, calmodulin. and metal-chelate resins, respectively. FLAG, c-mvc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolvtic cleavage site located between the HCSRP encoding sequence and the heterologous protein sequence. so that HCSRP may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention. synthesis of radiolabeled HCSRP may be achieved in vitro using the TNT rabbit reticulocvte iysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operable associated with the T7. T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor. for example, 'SS-methionine.
Fragments of HCSRP may be produced not only by recombinant means. but also by direct peptide synthesis using solid-phase techniques. (See, e.g.. Creiehton. su ra pp. »-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved. for example, using the ABI 431A peptide synthesizer (Perkin-Elmer).
Various fragments of HCSRP may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs. exists between regions of HCSRP and human cell surface receptor proteins. In addition, the expression of HCSRP is closely associated with lung, thymus. bladder. seminal vesicle, and penile tissues, with rheumatoid arthritis, and with inflammation, cancer, and the nervous system. Therefore.
HCSRP appears to play a role in cell proliferative disorders, immune system disorders, infections, and neuronal disorders. In IS the treatment of disorders associated with increased HCSRP expression or activity, it is desirable to decrease the expression or activity of HCSRP. In the treatment of disorders associated with decreased HCSRP expression or activity, it is desirable to increase the expression or activity of HCSRP.
Therefore, in one embodiment, HCSRP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HGSRP. Examples of such disorders include. but are not limited to, a cell proiiferative disorder such as actinic keratosis. arteriosclerosis, atherosclerosis, bursitis.
cirrhosis, hepatitis. mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria.
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia. lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma. and, in particular. cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid. penis, prostate, salivary glands. skin, spleen, testis, thymus, thyroid, and uterus: an immune system disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome. allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis. asthma. atherosclerosis. autoimmune hemolytic anemia.
autoimmune thyroiditis, bronchitis, bursitis, cholecystitis. cirrhosis, contact dermatitis. Crohn's disease. atopic dermatitis, dermatomyositis, diabetes mellitus. emphysema. erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis. Goodpasture's syndrome, gout, Graves' disease.
Hashimoto's thyroiditis, paroxysmal nocturnal hemoelobinuria. hepatitis, hypereosinophilia. irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD). multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myefofibrosis, osteoarthritis, osteoporosis, pancreatitis. polycvthemia vera. polymyositis. psoriasis.
Reiter's syndrome, rheumatoid arthritis. scleroderma. Sjogren's syndrome. systemic anaphylaxis, systemic lupus ennhematosus.
S systemic sclerosis, primary thrombocythemia. thrombocytopenic purpura.
ulcerative colitis, uveitis.
Werner syndrome. complications of cancer. hemodialysis. and extracorporeal circulation. trauma, and hematopoietic cancer including lymphoma. leukemia, and myeloma; an infection caused by a viral agent classified as adenovirus. arenavirus, bunyavirus, calicivirus, coronavirus. filovirus, hepadnavirus, herpesvirus. flavivirus. orthomyxovirus, parvovirus, papovavirus, paramyxovirus.
picornavirus, poxvirus, reovirus, retrovirus. rhabdovirus, or togavirus: an infection caused by a bacteria! agent classified as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium, clostridium, meningococcus. gonococcus, listeria, moraxella, kingella.
haemophilus, legionella, bordetella. gram-negative enterobacterium including shigella. salmonella. or campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces, l5 mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma: an infection caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other fungal agents causing various mycoses: an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania.
trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematodes such as trichinella, intestinal nematodes such as ascaris, lymphatic filarial nematodes, trematodes such as schistosoma, or cestrodes such as tapeworm: and a neuronal disorder such as akathesia.
Alzheimer's disease, amnesia. amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms. dementia, depression, diabetic neuropathy, Down's syndrome, tardive dyskinesia, dystonias. epilepsy, Huntington's disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's disorder.
In another embodiment, a vector capable of expressing HCSRP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HCSRP including, but not limited to. those described above.
In a further embodiment. a pharmaceutical composition comprising a substantially purified HCSRP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HCSRP including, but not limited to. those provided above.
In still another embodiment, an agonist which modulates the activity of HCSRP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HCSRP including, but not limited to, those fisted above.
In a further embodiment. an antagonist of HCSRP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HCSRP.
Examples of such disorders include, but are not limited to. those cell proliferative disorders.
immune system disorders.
infections, and neuronal disorders described above. In one aspect, an antibody which specifically binds HCSRP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express HCSRP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HCSRP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HCSRP including, but not limited to, those described above.
fn other embodiments. any of the proteins, antagonists, antibodies. agonists, complementary sequences. or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made I S by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach. one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of HCSRP may be produced using methods which are generally known in the art. In particular, purified HCSRP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HCSRP.
Antibodies to HCSRP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal. monoclonal. chimeric, and single chain antibodies, Fab fragments. and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
For the production of antibodies. various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HCSRP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to. Freund's. mineral eels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Conrnebacterium narvum are especially preferable.
It is preferred that the oligopeptides, peptides. or fragments used to induce antibodies to HCSRP have an amino acid sequence consisting of at least about ~ amino acids.
and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides. or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence ofa small. naturally occurring molecule. Short stretches of HCSRP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to HCSRP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
These include, but are not limited to. the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g.. Kohler, G. et al. ( 1975) Nature 256:495-497: Kozbor, D. et al. ( 1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. ( 1984) Mol. Cell Biol. 62:109-120.) In addition. techniques developed for the production of "chimeric antibodies."
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. ( 1984) Proc.
IS Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce HCSRP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. ( 1991 ) Proc. Natl. Acad. Sci. USA $8:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. ( 1989) Proc.
Natl. Acad. Sci. USA
86:3833-3837; Winter, G. et al. ( 1991 ) Nature 349:293-299.) Antibody fragments which contain specific binding sites for HCSRP may also be generated.
For example, such fragments inctude, but are not limited to, F(ab')= fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse. W.D.
et al. ( 1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between HCSRP and its specific antibody. A two-site. monoclonal-based immunoassay utilizing monoclonal antibodies reactive to iwo non-interfering HCSRP epitopes is Generally used. but a competitive binding assay may also be employed (Pound. supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassav techniques may be used to assess the affinity of antibodies for HCSRP. Affnity is expressed as an association constant, K,, which is defined as the molar concentration of HCSRP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
The K, determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple HCSRP epitopes, represents the average affinity, or avidity, of the antibodies for HCSRP. The K, determined for a preparation of monoclonal antibodies, which are monospecific for a particular HCSRP epitope, represents a true measure of affinity. High-affinity antibody preparations with K, ranging from about 10° to 10'= Llmole are preferred for use in immunoassays in which the HCSRP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in I S immunopurification and similar procedures which ultimately require dissociation of HCSRP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies.
Volume t: A Practical Approach, IRL Press, Washington, DC; Liddell, J.E. and Cryer, A. ( 1991 ) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least I-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of HCSRP-antibody complexes. Procedures for evaluating antibody specificity. titer, and avidity, and guidelines for antibody quality and usage in various applications. are generally available. (See, e.g., Catty, supra, and Coligan et al. s_~ra.}
In another embodiment of the invention. the polynucleotides encoding HCSRP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding EICSRP may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding HCSRP. Thus, complementary molecules or fragments may be used to modulate HCSRP activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HCSRP.

Expression vectors derived from retroviruses, adenoviruses. or herpes or vaccinia viruses. or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding HCSRP. (See. e.g., Sambrook, supra: Ausubel. 1995, supra.) Genes encoding HCSRP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide. or fragment thereof, encoding HCSRP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more . with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
As mentioned above. modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, S', or regulatory regions of the gene encoding HCSRP. Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may be employed.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E.
et al. (1994) in Huber, B.E. and B.I. Carr. Molecular and Immunolocic Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes. enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HCSRP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified. short RNA sequences of between 1 S and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding HCSRP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerise promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule. or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine.
l5 and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat.
Biotechnol. 15:162-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans. dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of HCSRP, antibodies to HCSRP, and mimetics, agonists. antagonists, or inhibitors of HCSRP. The compositions may be administered alone or in combination with at least one other agent.
such as a stabilizing compound. which may be administered in any sterile, biocompatible pharmaceutical carrier includins, but not limited to, saline. buffered saline. dextrose, and water. The compositions may be administered to a patient alone. or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to. oral. intravenous, intramuscular. infra-arterial.
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal. intranasal, enteral. topical, sublingual, or rectal means.
In addition to the active ingredients. these pharmaceutical compositions may contain suitable S pharmaceutically-acceptable carriers comprising excipients and awiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
Further details on techniques for formulation and administration may be found in the latest edition of Remineton's Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions. and the like. for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after IS grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants;
cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide. lacquer.solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin. as well as soft, sealed capsules made of gelatin and a coating. such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate. and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution. or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose. sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils. such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate. triglycerides, or tiposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular~barrier to be permeated are used in the for~rrtulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping. or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with many IS acids, including but not limited to, hydrochloric. sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0. I % to 2% sucrose, and 2%
to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of HCSRP, such labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats. rabbits, dogs, or pigs.
An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example HCSRP or fragments thereof, antibodies of HCSRP. and agonists, antagonists or inhibitors of HCSRP. which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDa° (the dose therapeutically effective in ~0%
of the population) or LDt° (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index. which can be expressed as the LDyEDi° ratio.
Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDa° with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient. and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject. time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 I S days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucieotides or polypeptides will be specific to particular cells, conditions. locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind HCSRP may be used for the diagnosis of disorders characterized by expression of HCSRP, or in assays to monitor patients being treated with HCSRP or agonists, antagonists, or inhibitors of HCSRP.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for HCSRP include methods which utilize the antibody and a label to detect HCSRP in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules. several of which are described above. are known in the art and may be used.
A variety of protocols for measuring HCSRP. including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of HCSRP expression.

Normal or standard values for HCSRP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example. human subjects, with antibody to HCSRP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods. such as photometric means. Quantities of HCSRP expressed in subject. control. and disease samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding HCSRP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of HCSRP
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of HCSRP, and to monitor regulation of HCSRP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences. including genomic sequences. encoding HCSRP or closely related molecules may be used to identify nucleic acid sequences which encode HCSRP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding HCSRP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50%
sequence identity to any of the HCSRP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:14-26 or from genomic sequences including promoters, enhancers, and introns of the HCSRP
gene.
Means for producing specific hybridization probes for DNAs encoding HCSRP
include the cloning of polynucleotide sequences encoding HCSRP or HCSRP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art. are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as'=P or'sS, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. and the like.
Polynucleotide sequences encoding HCSRP may be used for the diagnosis of disorders associated with expression of HCSRP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis.
hepatitis. mixed connective tissue disease (MCTD), myelofibrosis. paroxysmal nocturnal al hemogiobinuria. potycyhemia vera, psoriasis. primary thrombocythemia, and cancers including adenocarcinoma. leukemia, lymphoma. melanoma, myeloma. sarcoma, teratocarcinoma. and. in particular, cancers of the adrenal gland, bladder. bone. bone marrow, brain, breast. cervix, gall bladder. ganglia, gastrointestinal tract, heart. kidney, liver. lung, muscle.
ovary. pancreas, parathyroid, penis, prostate, salivary glands, skin. spleen. testis. thymus, thyroid, and uterus; an immune system disorder such as inflammation, actinic keratosis. acquired immunodeficiency syndrome (AIDS), Addison~s disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia. arteriosclerosis, asthma, atheroscierosis. autoimmune hemolytic anemia, autoimmune thyroiditis. bronchitis. bursitis, cholecystitis, cirrhosis. contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome. gout, Graves' disease, Hashimoto's thyroiditis. paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome. episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, IS myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, poiycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative colitis. uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeioma; an infection caused by a viral agent classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramvxovirus. picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus;
an infection caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus, legionella, bordetella, gram-negative enterobacterium including shigella, salmonella, or campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; an infection caused by a fungal agent classified as aspergillus. blastomyces, dermatophvtes. cryptococcus, coccidioides, malasezzia, histoplasma, or other fungal agents causing various mycoses; an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematodes such as trichinella, intestinal nematodes such as ascaris, lymphatic filarial nematodes, trematodes such as schistosoma, or cestrodes such as tapeworm: and a neuronal disorder such as akathesia. Alzheimer's disease. amnesia, amyotrophic lateral sclerosis. bipolar disorder, catatonia. cerebral neoplasms. dementia. depression, diabetic neuropathy.
Down's syndrome. tardive dyskinesia, dystonias, epilepsy, Huntington's disease, peripheral neuropathy.
multiple sclerosis, neurofibromatosis. Parkinson's disease, paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's disorder. The polynucleotide sequences encoding HCSRP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies: in PCR
technologies: in dipstick, pin, and multiformat ELISA-like assays: and in microarrays utilizing fluids or tissues from patients to detect altered HCSRP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect. the nucleotide sequences encoding HCSRP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding HCSRP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period. the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding HCSRP in the IS sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials. or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of HCSRP, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human. with a sequence, or a fragment thereof, encoding HCSRP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values. obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer. the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance .i3 of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding HCSRP may involve the use of PCR. These oligomers may be chemically synthesized. generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding HCSRP. or a fragment of a polynucleotide complementary to the polynucleotide encoding HCSRP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or !0 quantification ofclosely related DNA or RNA sequences. .
Methods which may also be used to quantify the expression of HCSRP include radiolabeling or biotinylating nucleotides. coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. ( 1993) J. Immunol. Methods I
X9:235-244; Duplaa. C.
et al. ( 1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be IS accelerated by running the assay in a high-throughput format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The microarray 20 can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used. and analyzed using methods known in the art. (See, e.g., 25 Brennan, T.M. et al. ( 1995) U.S. Patent No. 5.474,796; Schena, M. et al. ( 1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/2511 16;
Shalon, D, et al.
( 1995) PCT application W095/3550.i: Heller, R.A. et al. ( 1997) Proc. Natl.
Acad. Sci. USA 94:2150 2155; and Heller. M.J. et al. ( 1997) U.S. Patent No. 5,605,662.) In another embodiment of the invention, nucleic acid sequences encoding HCSRP
may be 30 used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g.. human artificial chromosomes (HACs), yeast artificial chromosomes (YACs}, bacterial artificial chromosomes (BACs), bacterial P 1 constructions. or single chromosome cDNA libraries. (See. e.g., Harrington, J.J. et al. ( 1997) Nat.
Genet. 15:345-355; Price, 4d C.M. ( 1993) Blood Rev. 7:127-134: and Trask. B.J. ( 1991 ) Trends Genet.
7:149-154.) Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Uirich, et at. ( 1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding HCSRP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder. may help define the region of DNA
associated with that disorder.
The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
t0 In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping.
This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 I q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. ( 1988) Nature 336:577-580.) The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, HCSRP, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between HCSRP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysers, et al. (1984) PCT
application W084/03564.) In this method. large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with HCSRP.
or fragments thereof, and washed. Bound HCSRP is then detected by methods well known in the art.
Purified HCSRP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
~t5 In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding HCSRP specifically compete with a test compound for binding HCSRP.
In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with HCSRP.
In additional embodiments, the nucleotide sequences which encode HCSRP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, in particular U.S. Ser. No. [Attorney Docket No. PF-0636 P, filed November 12, 1998), U.S. Ser. No.
[Attorney Docket No. PF-0650 P, filed December 7, 1998), and U.S. Ser. No.
60/123,404 are hereby 2o expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the Iysates with either isopropanoi or sodium acetate and ethanol. or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Ahernatively, RNA was isolated directly from tissue lysates using other RNA isolation kits. e.g., the POLY(A)PURE mRNA
.l6 WO 00/28032 PCTlUS99/26742 purification kit (Ambion. Austin TX).
In some cases. Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise. cDNA was synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997. supra, units ~. I-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000. SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid. e.g., PBLUESCRIPT plasmid (Stratagene). PSPORT1 plasmid (Life Technologies). or pINCY (Incyte Pharmaceuticals, Palo Alto CA). Recombinant plasmids were transformed into competent E. colt cells including XLl-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa. DHIOB.
or IS ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Piasmids were recovered from host cells by in vivo excision using the UN1ZAP
vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega}; an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN.
Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCB in a 35 high-throughput format (Rao. V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fruorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki. Finland).
III. Sequencing and Analysis cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Bobbins Scientific) or the MICROLAB ?200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared .l7 using rea~~ents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM B1GDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 37; or 377 sequencing system (Perkin-Elmer) in conjunction with standard ABl protocols and base calling software: or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, su ra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example V.
The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art. Table ~ summarizes the tools. programs. and algorithms used and provides applicable descriptions. references, and threshold parameters. The first column of Table ~ shows the tools, programs. and algorithms used, the second column provides brief descriptions thereof, the third I S column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences). Sequences were analyzed using MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software (DNASTAR). Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
The polynucleotide sequences were validated by removing vector. linker, and polyA
sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GenBank primate. rodent.
mammalian. vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO. PRODOM, and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length poiynucleotide sequences using programs based on Phred, Phrap, and Consed. and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length polynucleotide sequences were translated to derive the corresponding full length amino acid sequences. and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above). SwissProt.
BLOCKS, PRINTS, .i8 DOMO. PRODOM. Prosite. and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM is a probabilistic approach which analyzes consensus primary structures of gene families. (See. e.~;.. Eddy, S.R. ( 1996) Curr. Opin. Struct. Biol. 6:361-36~.) The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:14-26. Fragments from about 20 to about =1000 nucleotides which are useful in hybridization and amplification technologies were described in The invention section above.
IV. Northern Analysis Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte Pharmaceuticals). This IS analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
seauence identity x % maximum BLAST score The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1 % to 2% error, and, with a product score of 70, the match will be exact.
Similar molecules are usually identified by selecting those which show product scores between I ~
and 40, although lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding HCSRP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/irnmune, musculoskeletal, nervous, reproductive, and urologic. The disease/condition categories included cancer, infEammation, trauma, cell proliferation. neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.

V. Extension of HCSRP Encoding Polynucleotides The full length nucleic acid sequences of SEQ 1D N0:14-26 were produced by extension of an appropriate fragment of the full length molecule using oli~;onucleotide primers designed from this fragment. One primer was synthesized to initiate ~' extension of the known fragment. and the other primer. to initiate ~' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences). or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research.
Ine.). The reaction mix contained DNA template. 200 nmol of each primer. reaction buffer containing Mg-'', (NH,),SO.,, IS and p-mercaptoethanol. Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step I
: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min:
Step 5: Steps 2. 3, and 4 repeated 20 times;
Step 6: 68°C, S min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN: Molecular Probes, Eugene OR) dissolved in 1 X TE
and 0.5 Itl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan lI
(Labsystems Oy, Helsinki. Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A ~ ul to 10 ul aliquot of the reaction mixture was analyzed by electrophoresis on a I % agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised. and agar digested with Agar ACE
(Promega). Extended clones were relegated using T4 ligase (New England Biolabs. Beverly MA) into pUC 18 vector (Amersham Pharmacia 8iotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs. and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media. individual colonies were picked and cultured overnight at 37'C in 384-well plates in LB/2x Garb liquid media.
The cells were lysed. and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step I: 94°C, 3 min: Step?: 94°C, IS sec; Step 3:
60°C, t min: Step 4: 72°C. ? min;
Step ~: steps ~. 3. and 4 repeated 29 times: Step 6: 72°C, 5 min: Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulfoxide ( l :2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI

BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID NO: l4-26 are used to obtain ~' regulatory sequences using the procedure above, oligonucleotides designed for such extension. and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:14-26 are employed to screen cDNAs.
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about ?0 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, ?~0 uCi of [y-'=P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
An aliquot containing 10'counts per minute ofthe labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases:
Ase I. Bgl II, Eco RI, Pst !, Xba I, or Pvu 11 (DuPoni NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nvtran Plus. Schleicher & Schuell. Durham NH). Hybridization is carried out for t6 hours at 40°C. To remove nonspecific signals. blots are sequentially washed at room temperature under conditions of up to, for example. 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imagin~z means and SI

compared.
VII. Microarravs A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.~z.. Baldeschweiler, supra.) An array analogous to a dot or slot blot may also be used to arrange and lint: elements to the surface of a substrate using thermal.
UV, chemical, or mechanical bonding procedures. A typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
Full-length cDNAs, Expressed Sequence Tags (SSTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides Sequences complementary to the HCSRP-encoding sequences. or any parts thereof, are used to detect, decrease. or inhibit expression_of naturally occurring HCSRP.
Although use of oligonucleotides comprising from about 1 ~ to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucieotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of HCSRP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the HCSRP-encoding transcript.
IX. Expression of HCSRP
Expression and purification of HCSRP is achieved using bacterial or virus-based expression systems. For expression of HCSRP in bacteria. cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs hish levels of cDNA

transcription. Examples of such promoters include. but are not limited to, the ~rp-lcrc (mc) hybrid promoter and the T~ or T7 bacteriophage promoter in conjunction with the luc operator res~ulatorv element. Recombinant vectors are transformed into suitable bacterial hosts, e.g.. BL21(DE3).
Antibiotic resistant bacteria express HCSRP upon induction with isopropyl beta-D-~ thiogalactopyranoside (1PTG). Expression ofHCSRP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autosraphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding HCSRP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera frutLperda (Sf~) insect cells in most cases. or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-32'_'7: Sandig, V. et al.
(1996) Hum. Gene Ther.
7: I 93 7-1945. ) In most expression systems. HCSRP is synthesized as a fusion protein with.
e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from HCSRP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues. enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel ( 1995, su ra, ch. 10 and 16). Purified HCSRP obtained by these methods can be used directly in the following activity assay.
X. Demonstration of HCSRP Activity An assay for HCSRP activity measures the expression of HCSRP on the cell surface. cDNA
encoding HCSRP is transfected into an appropriate mammalian cell line. Cell surface proteins are labeled with biotin as described (de la Fuente. M. A. et al. ( 1997) Blood 90:2398-2405).
Immunoprecipitations are performed using HCSRP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of HCSRP
expressed on the cell surface.

An alternative assay for HCSRP activim is based on a prototypical assay for ligand/receptor-mediated modulation of cell proliferation. This assay measures the amount of newly synthesized DNA in Swiss mouse 3T3 cells expressing HCSRP. An appropriate mammalian expression vector containing cDNA encoding HCSRP is added to quiescent 3T3 cultured cells using transfection methods well known in the art. The transfected cells are incubated in the presence of ['H)thymidine and varying amounts of HCSRP ligand. Incorporation of ['H]thymidine into acid-precipitable DNA is measured over an appropriate time interval using a tritium radioisotope counter, and the amount incorporated is directly proportional to the amount of newly synthesized DNA.
A linear dose-response curve over at least a hundred-fold HCSRP ligand concentration range is indicative of receptor activiy. One unit of activity per milliliter is defined as the concentration of HCSRP
producing a 50% response level. where 100% represents maxima) incorporation of ['H]thymidine into acid-precipitable DNA. (McKay. I. and Leigh, I., eds. (1993) Growth Factors: A
Practical Approach, Oxford University Press. New York, NY, p. 73.) XI. Functional Assays HCSRP function is assessed by expressing the sequences encoding HCSRP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.
I (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-l0 ug of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line. using either liposome formulations or electroporation. 1-2 ~g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green 35 Fluorescent Protein (GFP: Clontech), CD64, or a CD64-GFP fusion protein.
Flow cyrtometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA
with propidium iodide; changes in cell size and granularity as measured by fonvard light scatter and 90 degree side Light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake: alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies: and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cyometry are discussed in Ormerod. Mt.G. ( 1994) Flow CWOmctrv~. Oxford.
New York NY.
The intluence of HCSRP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding HCSRP and either CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding HCSRP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XII. Production of HCSRP Specific Antibodies HCSRP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. ( 1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the HCSRP amino acid sequence is analyzed using LASERGENE
software I S (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 1 1.) Typically, oligopeptides of about 1 ~ residues in length are synthesized using an ABI 431 A
peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995. supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-HCSRP activii by, for example, binding the peptide or HCSRP to a substrate.
blocking with 1 35 BSA, reacting with rabbit antisera. washing, and reacting with radio-iodinated goat anti-rabbit lgG.
XIII. Purification of Naturally Occurring HCSRP Using Specific Antibodies Naturally occurring or recombinant HCSRP is substantially purified by immunoaffinity chromatography using antibodies specific for HCSRP. An immunoaffinity column is constructed by covalently coupling anti-HCSRP antibody to an activated chromatographic resin.
such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing HCSRP are passed over the immunoaffinity column. and the column is washed under conditions that allow the preferential absorbance of HCSRP (e.g..
high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/HCSRP binding (e.~~.. a buffer of pH ~ to pH 3. or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HCSRP is collected.
XIV. Identification of Molecules Which Interact with HCSRP
HCSRP. or biologically active fragments thereof. are labeled with'='I Bolton-Hunter reagent.
(See, e.g.. Bolton A.E. and W.M. Hunter ( 1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HCSRP, washed, and any wells with labeled HCSRP complex are assayed. Data obtained using different concentrations of HCSRP are used to calculate values for the number, affinity, and association of HCSRP with the candidate molecules.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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a n o. a. u° c°'n SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom CORLEY, Neil C.
GUEGLER, Karl J.
YUE, Henry BAUGHN, Mariah R.
LAL, Preeti HILLMAN, Jennifer L.
BANDMAN, Olga AZIMZAI, Yalda AU-YOUNG, Janice <120> HUMAN CELL SURFACE RECEPTOR PROTEINS
<130> PF-0636 PCT
<140> To Be Assigned <141> Herewith <150> 09/121,280; unassigned; 09/206,647; unassigned; 60/123,404 <151> 1998-11-12; 1998-11-I2; 1998-12-07; 1998-12-07; 1999-03-08 <160> 26 <170> PERL Program <210> 1 <211> 81 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2355971CD1 <400> 1 Met Gly Pro Arg Arg Leu Leu Leu Val Ala Ala Cys Phe Ser Leu Cys Gly Pro Leu Leu Ser Ala Arg Thr Arg Ala Arg Arg Pro Gly Glu Arg Cys Thr Gly Met Gly Cys Ala Gly Gly Gly Thr Pro Arg Gly Asp Cys Gly Gly His Cys Cys Asp Phe Ser Ser Pro Leu Pro Gln Phe Pro Pro Lys Ala Lys Leu Ala Phe Gly Leu Arg Ser Gly Val Phe Ser Ser His Val <210> 2 <211> 140 <212> PRT

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2917059CD1 <400> 2 Met Met Ala Gly Ile Arg Ala Leu Phe Met Tyr Leu Trp Leu Gln Leu Asp Trp Val Ser Arg Gly Glu Ser Val Gly Leu His Leu Pro Thr Leu Ser Val Gln Glu Gly Asp Asn Ser Ile Ile Asn Cys Ala Tyr Ser Asn Ser Ala Ser Asp Tyr Phe Ile Trp Tyr Lys Gln Glu Ser Gly Lys Gly Pro Gln Phe Ile Ile Asp Ile Arg Ser Asn Met Asp Lys Arg Gln Gly Gln Arg Val Thr Val Leu Leu Asn Lys Thr Val Lys His Leu Ser Leu Gln Ile Ala Ala Thr Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys Ala Glu Asn Thr His Cys Phe Pro Gly Ile Cys Asn His His Pro Asn Leu Arg Trp Glu Val Lys Gln His Pro Phe Pro Leu Gln <210> 3 <211> 358 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 538181CD1 <400> 3 Met Ala Trp Ala Leu Tyr Leu Ser.Leu Gly Val Leu Trp Val Ala Gln Met Leu Leu Ala Ala Gly Cys His Ala Ala Ala Ser Phe Glu Thr Leu Gln Cys Glu Gly Pro Val Cys Thr Glu Glu Ser Ser Cys His Thr Glu Asp Asp Leu Thr Asp Ala Arg Glu Ala Gly Phe Gln Val Lys Ala Tyr Thr Phe Ser Glu Pro Phe His Leu Ile Val Ser Tyr Asp Trp Leu Ile Leu Gln Gly Pro Ala Lys Pro Val Phe Glu Gly Asp Leu Leu Val Leu Arg Cys Gln Ala Trp Gln Asp Trp Pro Leu Thr Gln Val Thr Phe Tyr Arg Asp Gly Ser Ala Leu Gly Pro Pro Gly Pro Asn Arg Glu Phe Ser Ile Thr Val Val Gln Lys Ala Asp Ser Gly His Tyr His Cys Ser Gly Ile Phe Gln Ser Pro Gly Pro Gly Ile Pro Glu Thr Ala Ser Val Val Ala Ile Thr Val Gln Glu Leu Phe Pro Ala Pro Ile Leu Arg Ala Leu Pro Ser Ala Glu Pro Gln Ala Gly Gly Pro Met Thr Leu Ser Cys Gln Thr Lys Leu Pro Leu Gln Arg Ser Ala Ala Arg Leu Leu Phe Ser Phe Tyr Lys Asp Gly Arg Ile Val Gln Ser Arg Gly Leu Ser Ser Glu Phe Gln Ile Pro Thr Ala Ser Glu Asp His Ser Gly Ser Tyr Trp Cys Glu Ala Ala Thr Glu Asp Asn Gln Val Trp Lys Gln Ser Pro Gln Leu GIu Ile Arg Val Gln Gly Ala Ser Ser Ser Ala Ala Pro Pro Thr Leu Asn Pro Ala Pro Gln Lys Ser Ala Ala Pro Gly Thr Ala Pro Glu Glu Ala Pro Gly Pro Leu Pro Pro Pro Pro Thr Pro Ser Ser Glu Asp Pro Gly Phe Ser Ser Pro Leu Gly Met Pro Asp Pro His Leu Tyr His Gln Met Gly Leu Leu Leu Lys His Met Gln Asp Val Arg Val Leu Leu Gly His Leu Leu Met Glu Leu Arg Glu Leu Ser Gly His Arg Lys Pro Gly Thr Thr Lys Ala Thr Ala Glu <210> 4 <211> 201 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1368760CD1 <400> 4 Met Ala Arg Gln Lys Lys Met Gly Gln Ser Val Leu Arg Ala Val Phe Phe Leu Val Leu Gly Leu Leu Gly His Ser His Gly Gly Phe Pro Asn Thr Ile Ser Ile Gly Gly Leu Phe Met Arg Asn Thr Val Gln Glu His Ser Ala Phe Arg Phe Ala Val Gln Leu Tyr Asn Thr Asn Gln Asn Thr Thr Glu Lys Pro Phe His Leu Asn Tyr His Val Asp Leu Leu Asp Ser Ser Asn Ser Phe Ser Val Thr Asn Ala Phe Cys Ser Gln Phe Ser Arg Gly Val Tyr Ala Ile Phe Gly Phe Tyr Asp Gln Met Ser Met Asn Thr Leu Thr Ser Phe Cys Gly Ala Leu His Thr Ser Phe Val Thr Pro Ser Phe Pro Thr Asp Ala Asp Val Gln Phe Val Ile Gln Met Arg Pro Ala Leu Lys Gly Ala Ile Leu Ser Leu Leu Gly His Tyr Lys Trp Glu Lys Phe Val Tyr Leu Tyr Asp Thr Glu Arg Gly Lys Lys Arg His Leu Leu Cys Ser Leu Asp Ile His Val Ile Val Phe Lys Leu Pro Gln Leu Met Cys Pro Leu Leu Pro Ile Asn Lys Ile <210> 5 <211> 117 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1670669CD1 <400> 5 Met Asp His Leu Gly Ala Ser Leu Trp Pro Gln Val Gly Ser Leu Cys Leu Leu Leu Ala Gly Ala Ala Trp Ala Pro Pro Pro Asn Leu Pro Asp Pro Lys Phe Glu Ser Lys Ala Ala Leu Leu Ala Ala Arg Gly Pro Glu Glu Leu Leu Cys Phe Thr Glu Arg Val Gly Gly Z,eu Gly Met Ser His Gly Lys Leu Cys Arg Leu His Gln Ala Pro Thr Ala Arg Gly Gly Gly Ala Leu Leu Val Cys Ala Ala Tyr Arg Arg His Val Glu Leu Arg Ala Pro Arg Val Gly Arg His Ser Ser Leu Arg Arg Ser Ala Ile Ser Pro Cys His Pro His Gln <210> 6 <2I1> 455 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2851578CD1 4/~ ~

<400> 6 Met Glu Gly Gly Arg Ala Ala Gly Phe Pro Leu Ala Cys Lys Gln Glu His Arg Val Phe Leu Ser Gly Cys Glu Asn Ala Asp Glu Asn Pro Arg Met Leu Cys His Arg Gly Gly Gln Leu Ile Val Pro Ile Ile Pro Leu Cys Pro Glu His Ser Cys Arg Gly Arg Arg Leu Gln Asn Leu Leu Ser Gly Pro Trp Pro Lys Gln Pro Met Glu Leu His 65 70 . 75 Asn Leu Ser Ser Pro Ser Pro Ser Leu Ser Ser Ser Val Leu Pro Pro Ser Phe Ser Pro Ser Pro Ser Ser Ala Pro Ser Ala Phe Thr Thr Val Gly Gly Ser Ser Gly Gly Pro Cys His Pro Thr Ser Ser Ser Leu Val Ser Ala Phe Leu Ala Pro Ile Leu Ala Leu Glu Phe Val Leu Gly Leu Val Gly Asn Ser Leu Ala Leu Phe Ile Phe Cys Ile His Thr Arg Pro Trp Thr Ser Asn Thr Val Phe Leu Val Ser Leu Val Ala Ala Asp Phe Leu Leu Ile Ser Asn Leu Pro Leu Arg Val Asp Tyr Tyr Leu Leu His Glu Thr Trp Arg Phe Gly Ala Ala Ala Cys Lys Val Asn Leu Phe Met Leu Ser Thr Asn Arg Thr Ala Ser Val Val Phe Leu Thr Ala Ile Ala Leu Asn Arg Tyr Leu Lys Val Val Gln Pro His His Val Leu Ser Arg Ala Ser Val Gly Ala Ala Ala Arg Val Ala Gly Gly Leu Trp Val Gly Ile Leu Leu Leu Asn Gly His Leu Leu Leu Ser Thr Phe Ser Gly Pro Ser Cys Leu Ser Tyr Arg Val Gly Thr Lys Pro Ser Ala Ser Leu Arg Trp His Gln Ala Leu Tyr Leu Leu Glu Phe.Phe Leu Pro Leu Ala Leu Ile Leu Phe Ala Ile Val Ser Ile Gly Leu Thr Ile Arg Asn Arg Gly Leu Gly Gly Gln Ala Gly Pro Gln Arg Ala Met Arg Val Leu Ala Met Val Val Ala Val Tyr Thr Ile Cys Phe Leu Pro Ser Ile Ile Phe Gly Met Ala Ser Met Val Ala Phe Trp Leu Ser Ala Cys Arg Ser Leu Asp Leu Cys Thr Gln Leu Phe His Gly Ser Leu Ala Phe Thr Tyr Leu Asn Ser Val Leu Asp Pro Val Leu Tyr Cys Phe Ser Ser Pro Asn Phe Leu His Gln Ser Arg Ala Leu Leu Gly Leu Thr ~/? 3 Arg Gly Arg Gln Gly Pro Val Ser Asp Glu Ser Ser Tyr Gln Pro Ser Arg Gln Trp Arg Tyr Arg Glu Ala Ser Arg Lys Ala Glu Ala Ile Gly Lys Leu Lys Val Gln Gly Glu Val Ser Leu Glu Lys Glu Gly Ser Ser Gln Gly <210> 7 <211> 453 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3393757CD1 <400> 7 Met Gln Leu Asp Trp Asn Gln Ala Gln Lys Ser Gly Asp Pro Gly Pro Ser Val Val Gly Leu Val Ser Ile Pro Gly Met Gly Lys Leu Leu Ala Glu Ala Pro Leu Val Leu Glu Pro Glu Lys Gln Met Leu Leu His Glu Thr His Gln Gly Leu Leu Gln Asp Gly Ser Pro Ile Leu Leu Ser Asp Val Ile Ser Ala Phe Leu Ser Asn Asn Asp Thr Gln Asn Leu Ser Ser Pro Val Thr Phe Thr Phe Ser His Arg Ser Val Ile Pro Arg Gln Lys Val Leu Cys Val Phe Trp Glu His Gly Gln Asn Gly Cys Gly His Trp Ala Thr Thr Gly Cys Ser Thr Ile Gly Thr Arg Asp Thr Ser Thr Ile Cys Arg Cys Thr His Leu Ser Ser Phe Ala Val Leu Met Ala His Tyr Asp Val Gln Glu Glu Asp Pro Val Leu Thr Val Ile Thr Tyr Met Gly Leu Ser Val Ser Leu Leu Cys Leu Leu Leu Ala Ala Leu Thr Phe Leu Leu Cys Lys Ala Ile Gln Asn Thr Ser Thr Ser Leu His Leu Gln Leu Ser Leu Cys Leu Phe Leu Ala His Leu Leu Phe Leu Val Ala Ile Asp Gln Thr Gly His Lys Val Leu Cys Ser Ile Ile Ala Gly Thr Leu His Tyr Leu Tyr Leu Ala Thr Leu Thr Trp Met Leu Leu Glu Ala Leu Tyr Leu Phe Leu Thr Ala Arg Asn Leu Thr Val Val Asn Tyr Ser Ser Ile Asn Arg Phe Met Lys Lys Leu Met Phe Pro Val Gly Tyr Gly Val Pro Ala Val Thr Val Ala Ile Ser Ala Ala Ser Arg Pro His Leu Tyr Gly Thr Pro Ser Arg Cys Trp Leu Gln Pro Glu Lys Gly Phe Ile Trp Gly Phe Leu Gly Pro Val Cys Ala Ile Phe Ser Val Asn Leu Val Leu Phe Leu Val Thr Leu Trp Ile Leu Lys Asn Arg Leu Ser Ser Leu Asn Ser Glu Val Ser Thr Leu Arg Asn Thr Arg Met Leu Ala Phe Lys Ala Thr Ala Gln Leu Phe Ile Leu Gly Cys Thr Trp Cys Leu Gly Ile Leu Gln Val Gly Pro Ala Ala Arg Val Met Ala Tyr Leu Phe Thr Ile Ile Asn Ser Leu Gln Gly Val Phe Ile Phe Leu Val Tyr Cys Leu Leu Ser Gln Gln Val Arg Glu Gln Tyr Gly Lys Trp Ser Lys Gly Ile Arg Lys Leu Lys Thr Glu Ser Glu Met His Thr Leu Ser Ser Ser Ala Lys Ala Asp Thr Ser Lys Pro Ser Thr Val Arg Ser Arg Ile Ala Pro Glu His Phe Thr Asn Arg Pro Thr <210> 8 <211> 442 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 312256CD1 <400> 8 Met Ala Ser Val Val Leu Pro Ser Gly Ser Gln Cys Ala Ala Ala Ala Ala Ala Ala Ala Pro Pro Gly Leu Arg Leu Arg Leu Leu Leu Leu Leu Phe Ser Ala Ala Ala Leu Ile Pro Thr Gly Asp Gly Gln Asn Leu Phe Thr Lys Asp Val Thr Val Ile Glu Gly Glu Val Ala Thr Ile Ser Cys Gln Val Asn Lys Ser Asp Asp Ser Val Ile Gln Leu Leu Asn Pro Asn Arg Gln Thr Ile Tyr Phe Arg Asp Phe Arg Pro Leu Lys Asp Ser Arg Phe Gln Leu Leu Asn Phe Ser Ser Ser Glu Leu Lys Val Ser Leu Thr Asn Val Ser Ile Ser Asp Glu Gly Arg Tyr Phe Cys Gln Leu Tyr Thr Asp Pro Pro Gln Glu Ser Tyr Thr Thr Ile Thr Val Leu Val Pro Pro Arg Asn Leu Met Ile Asp Ile Gln Lys Asp Thr Ala Val Glu Gly Glu Glu Ile Glu Val Asn Cys Thr Ala Met Ala Ser Lys Pro Ala Thr Thr Ile Arg Trp Phe Lys Gly Asn Thr Glu Leu Lys Gly Lys Ser Glu Val Glu Glu Trp Ser Asp Met Tyr Thr Val Thr Ser Gln Leu Met Leu Lys Val His Lys Glu Asp Asp Gly Val Pro Val Ile Cys Gln Val Glu His Pro Ala Val Thr Gly Asn Leu Gln Thr Gln Arg Tyr Leu Glu Val Gln Tyr Lys Pro Gln Val His Ile Gln Met Thr Tyr Pro Leu Gln Gly Leu Thr Arg Glu Gly Asp Ala Leu Glu Leu Thr Cys Glu Ala Ile Gly Lys Pro Gln Pro Val Met Val Thr Trp Val Arg Val Asp Asp Glu Met Pro Gln His Ala Val Leu Ser Gly Pro Asn Leu Phe Ile Asn Asn Leu Asn Lys Thr Asp Asn Gly Thr Tyr Arg Cys Glu Ala Ser Asn Ile Val Gly Lys Ala His Ser Asp Tyr Met Leu Tyr Val Tyr Asp Pro Pro Thr Thr Ile Pro Pro Pro Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ile Leu Thr Ile Ile Thr Asp Ser Arg Ala Gly Glu Glu Gly Ser Ile Arg Ala Val Asp His Ala Val Ile Gly Gly Val Val Ala Val Val Val Phe Ala Met Leu Cys Leu Leu Ile Ile Leu Gly Arg Tyr Phe Ala Arg His Lys Gly Thr Tyr Phe Thr His Glu Ala Lys Gly.Ala Asp Asp Ala Ala Asp Ala Asp Thr Ala Ile Ile Asn Ala Glu Gly Gly Gln Asn Asn Ser Glu Glu Lys Lys Glu Tyr Phe Ile <210> 9 <211> 382 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1615704CD1 <400> 9 Met Asp Phe Leu Val Leu Phe Leu Phe Tyr Leu Ala Ser Val Leu Met Gly Leu Val Leu Ile Cys Val Cys Ser Lys Thr His Ser Leu Lys Gly Leu Ala Arg Gly Gly Ala Gln Ile Phe Ser Cys Ile Ile Pro Glu Cys Leu Gln Arg Ala Val His Gly Leu Leu His Tyr Leu Phe His Thr Arg Asn His Thr Phe Ile Val Leu His Leu Val Leu Gln Gly Met Val Tyr Thr Glu Tyr Thr Trp Glu Val Phe Gly Tyr Cys Gln Glu Leu Glu Leu Ser Leu His Tyr Leu Leu Leu Pro Tyr Leu Leu Leu Gly Val Asn Leu Phe Phe Phe Thr Leu Thr Cys Gly Thr Asn Pro Gly Ile Ile Thr Lys Ala Asn Glu Leu Leu Phe Leu His Val Tyr Glu Phe Asp Glu Val Met Phe Pro Lys Asn Val Arg Cys Ser Thr Cys Asp Leu Arg Lys Pro Ala Arg Ser Lys His Cys Ser Glu Cys Gly Ser Arg Asp Ser Ser Gly Thr Ser Asn Ser Thr Cys Val Gly Phe Val Cys Glu Gly Met Phe Pro Glu Ser Glu Ser Arg Ala Ser Ser Pro Pro Asp Met VaI Cys Val Thr Trp Cys Val His Arg Phe Asp His His Cys Val Trp Val Asn Asn Cys Ile Gly Ala Trp Asn Ile Arg Tyr Phe Leu Ile Tyr Val Leu Thr Leu Thr Ala Ser Ala Ala Thr Val Ala Ile Val Ser Thr Thr Phe Leu Val His Leu Val Val Met Ser Asp Leu Tyr Gln Glu Thr Tyr Ile Asp Asp Leu Gly His Leu His Val Met Asp Thr Val Phe Leu Ile Gln Tyr Leu Phe Leu Thr Phe Pro Arg.Ile Val Phe Met Leu Gly Phe Val Val Val Leu Ser Phe Leu Leu Gly Gly Tyr Leu Leu Phe Val Leu Tyr Leu Ala Ala Thr Asn Gln Thr Thr Asn Glu Trp Tyr Arg Gly Asp Trp Ala Trp Cys Gln Arg Cys Pro Leu Val Ala Trp Pro Pro Ser Ala GIu Pro Gln Val His Arg Asn Ile His Ser His Gly Leu Arg Ser Asn Leu Gln Glu Ile Phe Leu Pro Ala Phe Pro Cys His Glu Arg Lys Lys Gln Glu <210> 10 <211> 257 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1659465CD1 c400> 10 Met Ala Ser Lys Ile Gly Ser Arg Arg Trp Met Leu Gln Leu Ile Met Gln Leu Gly Ser Val Leu Leu Thr Arg Cys Pro Phe Trp Gly Cys Phe Ser Gln Leu Met Leu Tyr Ala Glu Arg Ala Glu Ala Arg Arg Lys Pro Asp Ile Pro Val Pro Tyr Leu Tyr Phe Asp Met Gly Ala Ala Val Leu Cys Ala Ser Phe Met Ser Phe Gly Val Lys Arg Arg Trp Phe Ala Leu Gly Ala Ala Leu Gln Leu Ala Ile Ser Thr Tyr Ala Ala Tyr Ile Gly Gly Tyr Val His Tyr Gly Asp Trp Leu Lys Val Arg Met Tyr Ser Arg Thr Val Ala Ile Ile Gly Gly Phe Leu Val Leu Ala Ser Gly Ala Gly Glu Leu Tyr Arg Arg Lys Pro Arg Ser Arg Ser Leu Gln Ser Thr Gly Gln Val Phe Leu Gly Ile Tyr Leu Ile Cys Val Ala Tyr Ser Leu Gln His Ser Lys Glu Asp Arg Leu Ala Tyr Leu Asn His Leu Pro Gly Gly Glu Leu Met Ile Gln Leu Phe Phe Val Leu Tyr Gly Ile Leu Ala Leu Ala Phe Leu Ser Gly Tyr Tyr Val Thr Leu Ala Ala Gln Ile Leu Ala Val Leu Leu Pro Pro Val Met Leu Leu Ile Asp Gly Asn Val Ala Tyr Trp His Asn Thr Arg Arg Val Glu Phe Trp Asn Gln Met Lys Leu Leu Gly Glu Ser Val Gly Ile Phe Gly Thr Ala Val Ile Leu Ala Thr Asp Gly <210> 11 c211> 697 c212> PRT
<213> Homo Sapiens c220>
<221> misc feature 1 ~/~ ~

<223> Incyte ID No: 2120743CD1 <400> 11 Met Cys Lys Ser Leu Arg Tyr Cys Phe Ser His Cys Leu Tyr Leu Ala Met Thr Arg Leu Glu Glu Val Asn Arg Glu Val Asn Met His Ser Ser Val Arg Tyr Leu Gly Tyr Leu Ala Arg Ile Asn Leu Leu Val Ala Ile Cys Leu Gly Leu Tyr Val Arg Trp Glu Lys Thr Ala Asn Ser Leu Ile Leu Val Ile Phe Ile Leu Gly Leu Phe Val Leu Gly Ile Ala Ser Ile Leu Tyr Tyr Tyr Phe Ser Met Glu Ala Ala Ser Leu Ser Leu Ser Asn Leu Trp Phe Gly Phe Leu Leu Gly Leu Leu Cys Phe Leu Asp Asn Ser Ser Phe Lys Asn Asp Val Lys Glu Glu Ser Thr Lys Tyr Leu Leu Leu Thr Ser Ile Val Leu Arg Ile Leu Cys Ser Leu Val Glu Arg Ile Ser Gly Tyr Val Arg His Arg Pro Thr Leu Leu Thr Thr Val Glu Phe Leu Glu Leu VaI Gly Phe Ala Ile Ala Ser Thr Thr Met Leu Val Glu Lys Ser Leu Ser Val Ile Leu Leu Val Val Ala Leu Ala Met Leu Ile Ile Asp Leu Arg Met Lys Ser Phe Leu Ala Ile Pro Asn Leu Val Ile Phe Ala Val Leu Leu Phe Phe Ser Ser Leu Glu Thr Pro Lys Asn Pro Ile Ala Phe Ala Cys Phe Phe Ile Cys Leu Ile Thr Asp Pro Phe Leu Asp Ile Tyr Phe Ser Gly Leu Ser Val Thr Glu Arg Trp Lys Pro Phe Leu Tyr Arg Gly Arg Ile Cys Arg Arg Leu Ser Val Val Phe Ala Gly Met Ile Glu Leu Thr Phe Phe. Ile Leu Ser Ala Phe Lys Leu Arg Asp Thr His Leu Trp Tyr Phe Val Ile Pro Gly Phe Ser Ile Phe Gly Ile Phe Trp Met Ile Cys His Ile Ile Phe Leu Leu Thr Leu Trp Gly Phe His Thr Lys Leu Asn Asp Cys His Lys Val Tyr Phe Thr His Arg Thr Asp Tyr Asn Ser Leu Asp Arg Ile Met Ala Ser Lys Gly Met Arg His Phe Cys Leu Ile Ser Glu Gln Leu Val Phe Phe Ser Leu Leu Ala Thr Ala Ile Leu Gly Ala Val Ser Trp Gln Pro Thr Asn Gly Ile Phe Leu Ser Met Phe Leu Ile Val Leu ~ 1/~~

Pro Leu Glu Ser Met Ala His Gly Leu Phe His Glu Leu Gly Asn Cys Leu Gly Gly Thr Ser Val Gly Tyr Ala Ile Val Ile Pro Thr Asn Phe Cys Ser Pro Asp Gly Gln Pro Thr Leu Leu Pro Pro Glu His Val Gln Glu Leu Asn Leu Arg Ser Thr Gly Met Leu Asn Ala Ile Gln Arg Phe Phe Ala Tyr His Met Ile Glu Thr Tyr Gly Cys Asp Tyr Ser Thr Ser Gly Leu Ser Phe Asp Thr Leu His Ser Lys Leu Lys Ala Phe Leu Glu Leu Arg Thr Val Asp Gly Pro Arg His Asp Thr Tyr Ile Leu Tyr Tyr Ser Gly His Thr His Gly Thr Gly Glu Trp Ala Leu Ala Gly Gly Asp Thr Leu Arg Leu Asp Thr Leu Ile Glu Trp Trp Arg Glu Lys Asn Gly Ser Phe Cys Ser Arg Leu Ile Ile Val Leu Asp Ser Glu Asn Ser Thr Pro Trp Val Lys Glu Val Arg Lys Ile Asn Asp Gln Tyr Ile Ala Val Gln Gly Ala Glu Leu Ile Lys Thr Val Asp Ile Glu Glu Ala Asp Pro Pro Gln Leu Gly Asp Phe Thr Lys Asp Trp Val Glu Tyr Asn Cys Asn Ser Ser Asn Asn Ile Cys Trp Thr Glu Lys Gly Arg Thr Val Lys Ala Val Tyr Gly Val Ser Lys Arg Trp Ser Asp Tyr Thr Leu His Leu Pro Thr Gly Ser Asp Val Ala Lys His Trp Met Leu His Phe Pro Arg Ile Thr Tyr Pro Leu Val His Leu Ala Asn Trp Leu Cys Gly Leu Asn Leu Phe Trp Ile Cys Lys Thr Cys Phe Arg Cys Leu Lys Arg Leu Lys Met Ser Trp Phe Leu Pro Thr Val Leu Asp Thr Gly Gln Gly Phe Lys Leu Val Lys Ser <210> 12 <211> 325 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3344986CD1 <400> 12 Met Ser Asp Ser Lys Glu Pro Arg Val Gln Gln Leu Gly Leu Leu 1 ~/~~

Gly Cys Leu Gly His Gly Ala Leu Val Leu Gln Leu Leu Ser Phe Met Leu Leu Ala Gly Val Leu Val Ala Ile Leu Val Gln Val Ser Lys Val Pro Ser Ser Leu Ser Gln Glu Gln Ser Glu Gln Asp Ala Ile Tyr Gln Asn Leu Thr Gln Leu Lys Ala Ala Val Gly Glu Leu Ser Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala Ala Val Gly Glu Leu Pro GIu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Asp Gln Ser Lys Gln Gln Gln Ile Tyr Gln Glu Leu Thr Asp Leu Lys Thr Ala Phe Glu Arg Leu Cys Arg His Cys Pro Lys Asp Trp Thr Phe Phe Gln Gly Asn Cys Tyr Phe Met Ser Asn Ser Gln Arg Asn Trp His Asp Ser Val Thr Ala Cys Gln Glu Val Arg Ala Gln Leu Val Val Ile Lys Thr Ala Glu Glu Gln Asn Phe Leu Gln Leu Gln Thr Ser Arg Ser Asn Arg Phe Ser Trp Met Gly Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp Gln Trp Val Asp Gly Ser Pro Leu Ser Pro Ser Phe Gln Arg Tyr Trp Asn Ser Gly Glu Pro Asn Asn Ser Gly Asn Glu Asp Cys Ala Glu Phe Ser Gly Ser Gly Trp Asn Asp Asn Arg Cys Asp Val Asp Asn Tyr Trp Ile Cys Lys Lys Pro Ala Ala Cys Phe Arg Asp Glu <210> 13 <211> 369 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3576503CD1 <400> 13 Met Lys Ser Pro Phe Tyr Arg Cys Gln Asn Thr Thr Ser Val Glu Lys Gly Asn Ser Ala Val Met Gly Gly Val Leu Phe Ser Thr Gly Leu Leu Gly Asn Leu Leu Ala Leu Gly Leu Leu Ala Arg Ser Gly Leu Gly Trp Cys Ser Arg Arg Pro Leu Arg Pro Leu Pro Ser Val Phe Tyr Met Leu Val Cys Gly Leu Thr Val Thr Asp Leu Leu Gly Lys Cys Leu Leu Ser Pro Val Val Leu Ala Ala Tyr Ala Gln Asn Arg Ser Leu Arg Val Leu Ala Pro Ala Leu Asp Asn Ser Leu Cys Gln Ala Phe Ala Phe Phe Met Ser Phe Phe Gly Leu Ser Ser Thr Leu Gln Leu Leu Ala Met Ala Leu Glu Cys Trp Leu Ser Leu Gly His Pro Phe Phe Tyr Arg Arg His Ile Thr Leu Arg Leu Gly Ala Leu Val Ala Pro Val Val Ser Ala Phe Ser Leu Ala Phe Cys AIa Leu Pro Phe Met Gly Phe Gly Lys Phe Val Gln Tyr Cys Pro Gly 170 1?5 180 Thr Trp Cys Phe Ile Gln Met Val His Glu Glu Gly Ser Leu Ser Val Leu Gly Tyr Ser Val Leu Tyr Ser Ser Leu Met Ala Leu Leu Val Leu Ala Thr Val Leu Cys Asn Leu Gly Ala Met Arg Asn Leu Tyr Ala Met His Arg Arg Leu Gln Arg His Pro Arg Ser Cys Thr Arg Asp Cys Ala Glu Pro Arg Ala Asp Gly Arg Glu Ala Ser Pro Gln Pro Leu Glu Glu Leu Asp His Leu Leu Leu Leu Ala Leu Met Thr Val Leu Phe Thr Met Cys Ser Leu Pro Val Ile Tyr Arg Ala Tyr Tyr Gly Ala Phe Lys Asp Val Lys Glu Lys Asn Arg Thr Ser Glu Glu Pro Glu Arg Pro Pro Ser Leu Ala Ile Ser Ile Cys Asp Val Asn Cys Gly Pro Leu Asp Ser Tyr His Phe Gln Ile Ser Ser Ile Ser Asp Ile Phe Ser Gln Asp Phe Ser Leu Asp Leu Leu Gly Thr Gly Ala Asp Ala Ala Ile Pro Leu Thr Trp Asn Pro Val Cys Asp Ser Val Phe His Ser Val Val Ser <210> 14 <211> 572 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2355971CB1 <400> 14 gcggagcagc ccgaggcggg gcagcctccc ggagcagcgc cgcgcagagc ccgggacaat 60 ggggccgcgg cggctgctgc tggtggccgc ctgcttcagt ctgtgcggcc cgctgttgtc 120 tgcccgcacc cgggcccgca ggccaggtga gagatgcacg ggaatggggt gcgcgggcgg 180 agggacgccg aggggagact gcgggggtca ctgttgcgac ttctcctcac ccctgcctca 240 gtttcctccg aaagccaaac tggcatttgg gctgagatct ggagtttttt ccagtcacgt 300 ttaggtgggg cgtgccaccc ccttcgttgg cacagccgat gcccctttgg actcgatctt 360 ggagggtgca gcccgcctgc aacggggtgt tggatatgga ggaagatgga gcggaagccc 420 ctgggggagc ctgcagtcct gcgttggaat tgtcaacaaa accgtttctt cccaaggacc 480 aacccccaaa aaggaaaagc ttctcaagtt ggtcccaacc aattaaacgt ttcggatctt 540 ttaaaaaaac aaaaaaccaa aaggggcggg cc 572 <210> 15 <211> 517 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2917059081 <400> 15 agaaaggaat acccgatgat ggaagtagct cttatggctg gagattgcag gtttatgact 60 gatcctattt gggaagaaca atgatggcag gcattcgagc tttatttatg tacttgtggc 120 tgcagctgga ctgggtgagc agaggagaga gtgtggggct gcatcttcct accctgagtg 180 tccaggaggg tgacaactct attatcaact gtgcttattc aaacagcgcc tcagactact 240 tcatttggta caagcaagaa tctggaaaag gtcctcaatt cattatagac attcgttcaa 300 atatggacaa aaggcaaggc caaagagtca ccgttttatt gaataagaca gtgaaacatc 360 tctctctgca aattgcagct actcaacctg gagactcagc tgtctacttt tgtgcagaga 420 atacacattg ctttccaggc atctgtaacc atcacccaaa cctgagatgg gaggtgaagc 480 agcatccctt tcctttgcaa taaattttag ttataga 517 <210> 16 <211> 2099 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 538181CB1 <400> 16 ggagacctaa acacagtcac catgaagctg ggctgtgtcc tcatggcctg ggccctctac 60 ctttcccttg gtgtgctctg ggtggcccag atgctactgg cagctggatg tcatgccgct 120 gccagttttg agacgctgca gtgtgaggga cctgtctgca ctgaggagag cagctgccac 180 acggaggatg acttgactga tgcaagggaa gctggcttcc aggtcaaggc ctacactttc 240 agtgaaccct tccacctgat tgtgtcctat gactggctga tcctccaagg tccagccaag 300 ccagtttttg aaggggacct gctggttctg cgctgccagg cctggcaaga ctggccactg 360 actcaggtga ccttctaccg agatggctca gctct3ggtc cccccgggcc taacagggaa 420 ttctccatca ccgtggtaca aaaggcagac agcgggcact accactgcag tggcatcttc 480 cagagccctg gtcctgggat cccagaaaca gcatctgttg tggctatcac agtccaagaa 540 ctgtttccag cgccaattct cagagctcta ccctcagctg aaccccaagc aggaggcccc 600 atgaccctga gttgtcagac aaagttgccc ctgcagaggt cagctgcccg cctcctcttc 660 tccttctaca aggatggaag gatagtgcaa agcagggggc tctcctcaga attccagatc 720 cccacagctt cagaagatca ctccgggtca tactggtgtg aggcagccac tgaggacaac 780 caagtttgga aacagagccc ccagctagag atcagagtgc agggtgcttc cagctctgct 840 gcacctccca cattgaatcc agctcctcag aaatcagctg ctccaggaac tgctcctgag 900 gaggcccctg ggcctctgcc tccgccgcca accccatctt ctgaggatcc aggcttttct 960 tctcctctgg ggatgccaga tcctcatctg tatcaccaga tgggccttct tctcaaacac 1020 atgcaggatg tgagagtcct cctcggtcac ctgctcatgg agttgaggga attatctggc 1080 caccggaagc ctgggaccac aaaggctact gctgaataga agtaaacagt tcatccatga 1140 tctcacttaa ccaccccaat aaatctgatt ctttattttc tcttcctgtc ctgcacatat 1200 gcataagtac ttttacaagt tgtcccagtg ttttgttaga ataatgtagt taggtgagtg 1260 taaataaatt tatataaagt gagaattaga gtttagctat aattgtgtat tctctcttaa 1320 cacaacagaa ttctgctgtc tagatcagga atttctatct gttatatcga ccagaatgtt 1380 gtgatttaaa gagaactaat ggaagtggat tgaatacagc agtctcaact gggggcaatt 1440 ttgcccccca gaggacattg ggaaatgttt ggagacattt tggtcattat acttgggggg 1500 ttgggggatg gtgggatgtg tgtgctactg gcatccagta aatagaagcc aggggtgccg 1560 ctaaacatcc tataatgcac agggcagtac cccacaacga aaaataatct ggcccaaaat 1620 gtcagttgta ctgagtttga gaaaccccag cctaatgaaa ccctaggtgt tgggctctgg 1680 aatgggactt tgtcccttct aattattatc tctttccagc ctcattcagc tattcttact 1740 gacataccag tctttagctg gtgctatggt ctgttcttta gttctagttt gtatcccctc 1800 aaaagccatt atgttgaaat cctaatcccc aaggtgatgg cattaagaag tgggcctttg 1860 ggaagtgatt agatcaggag tgcagagccc tcatgattag gattagtgcc cttatttaaa 1920 aaggccccag agagctaact cacccttcca ccatatgagg acgtggcaag aagatgacat 1980 gtatgagaac caaaaaacag tgtcgccaaa caccgactct gtcgttgcct tgatcttgaa 2040 cttccagcct ccagaactat gagaaataaa attctgttgt ttgtaagcta aaaaaaaaa 2099 <210> 17 <211> 770 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1368760CB1 <400> 17 agtgtggggt ggaaaggaag agtgagcgag agcaagttaa ggggaggggg tgtaagagcc 60 agcgaattct ttttcttttt ctattattat tttgacgact cctgagttgc gcccatgctc 120 ttgtcagctt cgttttaggc gtacatggcc aggcagaaga aaatggggca aagcgtgctc 180 cgggcggtct tctttttagt cctggggctt ttgggtcatt ctcacggagg attccccaac 240 accatcagca taggtggact tttcatgaga aacacagtgc aggagcacag cgctttccgc 300 tttgccgtgc agttatacaa caccaaccag aacaccaccg agaagccctt ccatttgaat 360 taccacgtag atctcttgga ttcctccaat agtttttccg tgacaaatgc tttctgctcc 420 cagttctcga gaggggtgta tgccatcttt ggattctatg accagatgtc aatgaacacc 480 ctgacctcct tctgtggggc cctgcacaca tcctttgtta cgcctagctt ccccactgac 540 gcagatgtgc agtttgtcat ccagatgcgc ccagccttga agggcgctat tctgagtctt 600 ctgggtcatt acaagtggga gaagtttgtg tacctctatg acacagaacg aggtaagaag 660 aggcacctgc tctgctcttt agatattcat gtaattgtgt tcaaacttcc tcagcttatg 720 tgccctttgc ttccaataaa taaaatctaa ttctgtttta aaaaaaaaaa 770 WO 00/28032 PCT/US99l26742 <210> 18 <211> 572 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1670669CB1 <400> 18 gcctggtcgg gaagggcctg gtcagctgcg tccggcggag gcagctgctg acccagctgt 60 ggactgtgcc ggtggcgggg gacggagggg caggagccct gggctccccg tgggcggggg 120 ctgtatcatg gaccacctcg gggcgtccct ctggccccag gtcggctccc tttgtctcct 180 gctcgctggg gccgcctggg cgcccccgcc taacctcccg gaccccaagt tcgagagcaa 240 agcggccttg ctggcggccc gggggcccga agagcttctg tgcttcaccg agcgggttgg 300 aggacttggg atgagccatg ggaagctgtg tcgcctgcac caggctccca cggcgcgtgg 360 tggcggtgcg cttctggtgt gcgctgccta caggcgacac gtcgagcttc gtgcccctag 420 agttgggcgt cacagcagcc tccggcgctc cgcgatatca ccgtgtcatc cacatcaatg 480 aagtagtgct ccgagacggc cccgtggggc tggtggcgcg ggttggctga cgagagcggg 540 cagctaggtg tgcgctggct cccggggggt gg 572 <210> 19 <211> 1795 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2851578CB1 <400> 19 ctcagtaccc cgaggcggat ggaaggaggg agggctgcag ggttcccctt ggcctgcaaa 60 caggaacaca gggtgtttct cagtggctgc gagaatgctg atgaaaaccc caggatgttg 120 tgtcaccgtg gtggccagct gatagtgcca atcatcccac tttgccctga gcactcctgc 180 aggggtagaa gactccagaa ccttctctca ggcccatggc ccaagcagcc catggaactt 240 cataacctga gctctccatc tccctctctc tcctcctctg ttctccctcc ctccttctct 300 ccctcaccct cctctgctcc ctctgccttt accactgtgg gggggtcctc tggagggccc 360 tgccacccca cctcttcctc gctggtgtct gccttcctgg caccaatcct ggccctggag 420 tttgtcctgg gcctggtggg gaacagtttg gccctcttca tcttctgcat ccacacgcgg 480 ccctggacct ccaacacggt gttcctggtc agcctggtgg ccgctgactt cctcctgatc 540 agcaacctgc ccctccgcgt ggactactac ctcctccatg agacctggcg ctttggggct 600 gctgcctgca aagtcaacct cttcatgctg tccaccaacc gcacggccag cgttgtcttc 660 ctcacagcca tcgcactcaa ccgctacctg aaggtggtgc agccccacca cgtgctgagc 720 cgtgcttccg tgggggcagc tgcccgggtg gccgggggac tctgggtggg catcctgctc 780 ctcaacgggc acctgctcct gagcaccttc tccggcccct cctgcctcag ctacagggtg 840 ggcacgaagc cctcggcctc gctccgctgg caccaggcac tgtacctgct ggagttcttc 900 ctgccactgg cgctcatcct ctttgctatt gtgagcattg ggctcaccat ccggaaccgt 960 ggtctgggcg ggcaggcagg cccgcagagg gccatgcgtg tgctggccat ggtggtggcc 1020 gtctacacca tctgcttctt gcccagcatc atctttggca tggcttccat ggtggctttc 1080 tggctgtccg cctgccgctc cctggacctc tgcacacagc tcttccatgg ctccctggcc 1140 ttcacctacc tcaacagtgt cctggacccc gtgctctact gcttctctag ccccaacttc 1200 ctccaccaga gccgggcctt gctgggcctc acgcggggcc ggcagggccc agtgagcgac 1260 gagagctcct accaaccctc caggcagtgg cgctaccggg aggcctctag gaaggcggag 1320 gccataggga agctgaaagt gcagggcgag gtctctctgg aaaaggaagg ctcctcccag 1380 ggctgagggc cagctgcagg gctgcagcgc tgtgggggta agggctgccg cgctctggcc 1440 tggagggaca aggccagcac acggtgcctc aaccaactgg acaagggatg gcggcagacc 1500 aggggccagg ccaaagcact ggcaggactc aggtgggtgg cagggagaga aacccaccta 1560 ggcctctcag tgtgtccagg atggcattcc cagaatgcag gggagagcag gatgccgggt 1620 ggaggagaca ggcaaggtgc cgttggcaca ccagctcaga caggggcctg cgcagctgca 1680 ggggacagac gccaatcact gtcacagcag agtcacctta gaaattggac agctgcatgt 1740 tctgtgctct ccagtttgtc ccttccaata ttaataaact tcccttttaa atata 1795 <210> 20 <211> 2053 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3393757CB1 <400> 20 cccaccctca tcggcctccc aaagtgctgg aattagaggc gtgatcaccg tgcccagccg 60 ccaatgccat cttcatcccc cagatagaca gtctctagga tctgttccct ggggctgagc 120 ggttggagtc ttcatgcggg ccctctggcc catggctcac taggtctgtg tccacatccc 180 tccagagcat cttacaggcg ctggatgagc tgctggaggc ccctggggac ctggagaccc 240 tgccccgctt acagcagcac tgtgtggcca gtcacctgct ggatggccta gaggatgtcc 300 tcagaggcct gagcaagaac ctttccaatg ggctgttgaa cttcagttat cctgcaggca 360 cagaattgtc cctggaggtg cagaagcaag tagacaggag tgtcaccttg agacagaatc 420 aggcagtgat gcagctcgac tggaatcagg cacagaaatc tggtgaccca ggcccttctg 480 tggtgggcct tgtctccatt ccagggatgg gcaagttgct ggctgaggcc cctctggtcc 540 tggaacctga gaagcagatg cttctgcatg agacacacca gggcttgctg caggacggct 600 cccccatcct gctctcagat gtgatctctg cctttctgag caacaacgac acccaaaacc 660 tcagctcccc agttaccttc accttctccc accgttcagt gatcccgaga cagaaggtgc 720 tctgtgtctt ctgggagcat ggccagaatg gatgtggtca ctgggccacc acaggctgca 780 gcacaatagg caccagagac accagcacca tctgccgttg cacccacctg agcagctttg 840 ccgtcctcat ggcccactac gatgtgcagg aggaggatcc cgtgctgact gtcatcacct 900 acatggggct gagcgtctct ctgctgtgcc tcctcctggc ggccctcact tttctcctgt 960 gtaaagccat ccagaacacc agcacctcac tgcatctgca gctctcgctc tgcctcttcc 1020 tggcccacct cctcttcctc gtggcaattg atcaaaccgg acacaaggtg ctgtgctcca 1080 tcatcgccgg taccttgcac tatctctacc tggccacctt gacctggatg ctgctggagg 1140 ccctgtacct cttcctcact gcacggaacc tgacggtggt caactactca agcatcaaca 1200 gattcatgaa gaagctcatg ttccctgtgg gctacggagt cccagctgtg acagtggcca 1260 tttctgcagc ctccaggcct cacctttatg gaacaccttc ccgctgctgg ctccaaccag 1320 aaaagggatt tatatggggc ttccttggac ctgtctgcgc catcttctct gtgaatttag 1380 ttctctttct ggtgactctc tggattttga aaaacagact ctcctccctc aatagtgaag 1440 tgtccaccct ccggaacaca aggatgctgg catttaaagc gacagctcag ctgttcatcc 1500 tgggctgca ;gtggtgtctg ggcatcttgc aggtgggtcc ggctgcccgg gtcatggcct 1560 acctcttcac catcatcaac agcctgcagg gtgtcttcat cttcctggtg tactgcctcc 1620 tcagccagca ggtccgggag caatatggga aatggtccaa agggatcagg aaattgaaaa 1680 ctgagtctga gatgcacaca ctctccagca gtgctaaggc tgacacctcc aaacccagca 1740 cggtaagatc acgcattgct ccagagcact tcactaaccg acccacctga ggagcatgtg 1800 cctatcacac aaggaaacct gggaatacag caggcaatgc cctagaaagg ctcgcatctg 1860 agtacgcctt gactcattaa ccattagcaa tgatctcagt ttaaatgttt ttttttaatc 1920 agtcatagcc tgtcatcccg gcatcactgt catcccagca tttgggaggc ctaggcaaga 1980 gggtcacctg aggccaggag tgcaagatga ccctgggcaa catagcaaga tcccatctct 2040 acaaaaaaaa aaa 2053 <210> 21 <211> 1500 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 312256CH1 <400> 21 caggtgcccg acatggcgag tgtagtgctg ccgagcggat cccagtgtgc ggcggcagcg 60 gcggcggcgg cgcctcccgg gctccggctc cggcttctgc tgttgctctt ctccgccgcg 120 gcactgatcc ccacaggtga tgggcagaat ctgtttacga aagacgtgac agtgatcgag 180 ggagaggttg cgaccatcag ttgccaagtc aataagagtg acgactctgt gattcagcta 240 ctgaatccca acaggcagac catttatttc agggacttca ggcctttgaa ggacagcagg 300 tttcagttgc tgaatttttc tagcagtgaa ctcaaagtat cattgacaaa cgtctcaatt 360 tctgatgaag gaagatactt ttgccagctc tataccgatc ccccacagga aagttacacc 420 accatcacag tcctggtccc accacgtaat ctgatgatcg atatccagaa agacactgcg 480 gtggaaggtg aggagattga agtcaactgc actgctatgg ccagcaagcc agccacgact 540 atcaggtggt tcaaagggaa cacagagcta aaaggcaaat cggaggtgga agagtggtca 600 gacatgtaca ctgtgaccag tcagctgatg ctgaaggtgc acaaggagga cgatggggtc 660 ccagtgatct gccaggtgga gcaccctgcg gtcactggaa acctgcagac ccagcggtat 720 ctagaagtac agtataagcc tcaagtgcac attcagatga cttatcctct acaaggctta 780 acccgggaag gggacgcgct tgagttaaca tgtgaagcca tcgggaagcc ccagcctgtg 840 atggtaactt gggtgagagt cgatgatgaa atgcctcaac acgccgtact gtctgggccc 900 aacctgttca tcaataacct aaacaaaaca gataatggta cataccgctg tgaagcttca 960 aacatagtgg ggaaagctca ctcggattat atgctgtatg tatacgatcc ccccacaact 1020 atccctcctc ccacaacaac caccaccacc accaccacca ccaccaccac catccttacc 1080 atcatcacag attcccgagc aggtgaagaa ggctcgatca gggcagtgga tcatgccgtg 1140 atcggtggcg tcgtggcggt ggtggtgttc gccatgctgt gcttgctcat cattctgggg 1200 cgctattttg ccagacataa aggtacatac ttcactcatg aagccaaagg agccgatgac 1260 gcagcagacg cagacacagc tataatcaat gcagaaggag gacagaacaa ctccgaagaa 1320 aagaaagagt acttcatcta gatcagcctt tttgtttcaa tgaggtgtcc aactggccct 1380 atttagatga taaagagaca gtgatattgg aacttgcgag aaattcgtgt gtttttttat 1440 gaatgggtgg aaaggtgtga gactgggaag gcttgggatt tgctgtgtaa aaaaaaaaaa 1500 <210> 22 <21I> 1449 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1615704CH1 <400> 22 gtcacgagcc cgcaggaagt ctcgtatcgc gcccgggagg cgccggagcc.cagcggctgg 60 cgccagatcc aggctcctgg aagaaccatg tccggcagct actggtcatg ccaggcacac 120 actgctgccc aagaggagct gctgtttgaa ttatctgtga atgttgggaa gaggaatgcc 180 agagctgccg gctgaaaatt acccaaccaa gagaaatctg caggatggac tttctggtcc 240 tcttcttgtt ctacctggct tcggtgctga tgggtcttgt tcttatctgc gtctgctcga 300 aaacccatag cttgaaaggc ctggccaggg gaggagcaca gatattttcc tgtataattc 360 cagaatgtct tcagagagcc gtgcatggat tgcttcatta ccttttccat acgagaaacc 420 acaccttcat tgtcctgcac ctggtcttgc aagggatggt ttatactgag tacacctggg 480 WO 00/28032 PCT/US99/26?42 aagtatttgg ctactgtcag gagctggagt tgtccttgca ttaccttctt ctgccctatc 540 tgctgctagg tgtaaacctg ttttttttca ccctgacttg tggaaccaat cctggcatta 600 taacaaaagc aaatgaatta ttatttcttc atgtttatga atttgatgaa gtgatgtttc 660 caaagaacgt gaggtgctct acttgtgatt taaggaaacc agctcgatcc aagcactgca 720 gtgagtgtgg ctctcgtgac tccagcggca cctccaacag cacatgtgtg ggcttcgtct 780 gtgagggaat gtttcctgaa tccgaaagca gagccagttc acccccagat atggtgtgtg 840 taacctggtg tgtgcaccgt ttcgaccatc actgtgtttg ggtgaacaac tgcatcgggg 900 cctggaacat caggtacttc ctcatctacg tcttgacctt gacggcctcg gctgccaccg 960 tcgccattgt gagcaccact tttctggtcc acttggtggt gatgtcagat ttataccagg 1020 agacttacat cgatgacctt ggacacctcc atgttatgga cacggtcttt cttattcagt 1080 acctgttcct gacttttcca cggattgtct tcatgctggg ctttgtcgtg gttctgagct 1140 tcctcctggg tggctacctg ttgtttgtcc tgtatctggc ggccaccaac cagactacta 1200 acgagtggta cagaggtgac tgggcctggt gccagcgttg tccccttgtg gcctggcctc 1260 cgtcagcaga gccccaagtc caccggaaca ttcactccca tgggcttcgg agcaaccttc 1320 aagagatctt tctacctgcc tttccatgtc atgagaggaa gaaacaagaa tgacaagtgt 1380 atgactgcct ttgagctgta gttcccgttt atttacacat gtggatcctc gttttccaaa 1440 aaaaaaaaa 1449 <210> 23 <211> 1587 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1659465CB1 <400> 23 tgtcccatcc ccgcgcctgc ttctctgact ggggtgaggc cgcagcggac tgccctttcc 60 caagatggcg tcgaagatag gttcgagacg gtggatgttg cagctgatca tgcagttggg 120 ttcggtgctg ctcacacgct gccccttttg gggctgcttc agccagctca tgctgtacgc~180 tgagagggct gaggcacgcc ggaagcccga catcccagtg ccttacctgt atttcgacat 240 gggggcagcc gtgctgtgcg ctagtttcat gtcctttggc gtgaagcggc gctggttcgc 300 gctgggggcc gcactccaat tggccattag cacctacgcc gcctacatcg ggggctacgt 360 ccactacggg gactggctga aggtccgtat gtactcgcgc acagttgcca tcatcggcgg 420 ctttcttgtg ttggccagcg gtgctgggga gctgtaccgc cggaaacctc gcagccgctc 480 cctgcagtcc accggccagg tgttcctggg tatctacctc atctgtgtgg cctactcact 540 gcagcacagc aaggaggacc ggctggcgta tctgaaccat ctcccaggag gggagctgat 600 gatccagctg ttcttcgtgc tgtatggcat cctggccctg gcctttctgt caggctacta 660 cgtgaccctc gctgcccaga tcctggctgt actgctgccc cctgtcatgc tgctcattga 720 tggcaatgtt gcttactggc acaacacgcg gcgtgttgag ttctggaacc agatgaagct 780 ccttggagag agtgtgggca tcttcggaac tgctgtcatc ctggccactg atggctgagt 840 tttatggcaa gaggctgaga tgggcacagg gagccactga gggtcaccct gccttcctcc 900 ttgctggccc agctgctgtt tatttatgct ttttggtctg tttgtttgat cttttgcttt 960 tttaaaattg ttttttgcag ttaagaggca gctcatttgt ccaaatttct gggctcagcg 1020 cttgggaggg caggagccct ggcactaatg ctgtacaggt ttttttcctg ttaggagagc 1080 tgaggccagc tgcccactga gtctcctgtc cctgagaagg gagtatggca gggctgggat 1140 gcggctactg agagtgggag agtgggagac agaggaagga agatggagat tggaagtgag 1200 caaatgtgaa aaattcctct ttgaacctgg cagatgcagc taggctctgc agtgctgttt 1260 ggagactgtg agagggagtg tgtgtgttga cacatgtgga tcaggcccag gaagggcaca 1320 ggggctgagc actacagaag tcacatgggt tctcagggta tgccaggggc agaaacagta 1380 ccggctctct gtcactcacc ttgagagtag agcagaccct gttctgctct gggctgtgaa 1440 ggggtggagc aggcagtggc cagctttgcc cttcctgctg tctctgtttc tagctccatg 1500 gttggcctgg tgggggtgga gttccctccc aaacaccaga ccacacagtc ctccaaaaat 1560 aaacatttta tatagaaaaa aaaaaaa <210> 24 <211> 3928 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2120743CB1 <400> 24 ccctcgagcc gtaccgtcgc ggatttcggc ggcggaaaca tggcggtcgc ggccgggccg 60 gtaacggaga aagtttacgc cgacactggc ctgtattagc gcgtatggcc tcgggccctc 120 gttccccaag gcgtgccgcc tccctgttct cagtcgcagg ctgaagcctt gtctgctctc 180 ctcctttttg gtttggtttt ggaactgact ccgagggttg ggagagcgcg ttggtggcga 240 cggccgagtc agatcactat aaacaaaatt tccacaagag aaaatgttga aataggagtt 300 gcggatacat tggatatact ggatgaaata caagcggtta attttgtaac gtgagggaaa 360 agcccacatt gctggttaca tgtgtaaatc actgcgttat tgctttagtc attgtctcta 420 tttagcaatg acaagactgg aagaagtaaa tagagaagtg aacatgcatt cttcagtgcg 480 gtatcttggc tatttagcca gaatcaattt attggttgct atatgcttag gtctatacgt 540 aagatgggaa aaaacagcaa attccttaat tttggtaatt tttattcttg gtctttttgt 600 tcttggaatc gccagcatac tctattacta tttttcaatg gaagcagcaa gtttaagtct 660 ctccaatctt tggtttggat tcttgcttgg cctcctatgt tttcttgata attcatcctt 720 taaaaatgat gtaaaagaag aatcaaccaa atatttgctt ctaacatcca tagtgttaag 780 gatattgtgc tctctggtgg agagaatttc tggttatgtc cgtcatcggc ccactttact 840 aaccacagtt gaatttctgg agcttgttgg atttgccatt gccagcacaa ctatgttggt 900 ggagaagtct ctgagtgtca ttttgcttgt tgtagctctg gctatgctga ttattgatct 960 gagaatgaaa tctttcttag ctattccaaa cttagttatt tttgcagttt tgttattttt 1020 ttcctcattg gaaactccca aaaatccgat tgcttttgcg tgttttttta tttgcctgat 1080 aactgatcct ttccttgaca tttattttag tggactttca gtaactgaaa gatggaaacc 1140 ctttttgtac cgtggaagaa tttgcagaag actttcagtc gtttttgctg gaatgattga 1200 gcttacattt tttattcttt ccgcattcaa acttagagac actcacctct ggtattttgt 1260 aatacctggc ttttccattt ttggaatttt ctggatgatt tgtcatatta tttttctttt 1320 aactctttgg ggattccata ccaaattaaa tgactgccat aaagtatatt ttactcacag 1380 gacagattac aatagccttg atagaatcat ggcatccaaa gggatgcgcc atttttgctt 1440 gatttcagag cagttggtgt tctttagtct tcttgcaaca gcgattttgg gagcagtttc 1500 ctggcagcca acaaatggaa ttttcttgag catgtttcta atcgttttgc cattggaatc 1560 catggctcat gggctcttcc atgaattggg taactgttta ggaggaacat ctgttggata 1620 tgctattgtg attcccacca acttctgcag tcctgatggt cagccaacac tgcttccccc 1680 agaacatgta caggagttaa atttgaggtc tactggcatg ctcaatgcta tccaaagatt 1740 ttttgcatat catatgattg agacctatgg atgtgactat tccacaagtg gactgtcatt 1800 tgatactctg cattccaaac taaaagcttt cctcgaactt cggacagtgg atggacccag 1860 acatgatacg tatattttgt attacagtgg gcacacccat ggtacaggag agtgggctct 1920 agcaggtgga gatacactac gccttgacac acttatagaa tggtggagag aaaagaatgg 1980 ttccttttgt tcccggctta ttatcgtatt agacagcgaa aattcaaccc cttgggtgaa 2040 agaagtgagg aaaattaatg accagtatat tgcagtgcaa ggagcagagt tgataaaaac 2100 agtagatatt gaagaagctg acccgccaca gctaggtgac tttacaaaag actgggtaga 2160 atataactgc aactccagta ataacatctg ctggactgaa aagggacgca cagtgaaagc 2220 agtatatggt gtgtcaaaac ggtggagtga ctacactctg catttgccaa cgggaagcga 2280 tgtggccaag cactggatgt tacactttcc tcgtattaca tatcccctag tgcatttggc 2340 aaattggtta tgcggtctga accttttttg gatctgcaaa acttgtttta ggtgcttgaa 2400 aagattaaaa atgagttggt ttcttcctac tgtgctggac acaggacaag gcttcaaact 2460 tgtcaaatct taatttggac cccaaagcgg gatattaata agcactcata ctaccaatta 2520 tcactaactt gccatttttt gtatgctgta tttttatttg tggaaaatac cttgctactt 2580 ctgtagctgc tctcactttg tcttttctta agtaattatg gtatatataa ggcgttggga 2640 aaaaacattt tataatgaaa gtatgtaggg agtcaaatgc ttactgtaaa tgcataagag 2700 acgttaaaaa taacactgca ctttcaggaa tgtttgctta tggtcctgat tagaaagaaa 2760 cagttgtcta tgctctgcaa tggtcaatga tgaattacta atgccttatt ttctaggcat 2820 ataataatag tttagagaat gtagaccaga taaatttgtt tactgtttta agaaaactac 2880 cagtttactt acagaagatt cttttttcca aacagtaggt ttcatccaag accatttgaa 2940 gaactgcaaa ctctttctct tagaaaagaa agagggcagc ctaaaataaa cgcaaaattt 3000 gcttatactc catcacattc agatgtcttg gttgtgactt attaccagtg tggcagagaa 3060 cccaagttac attttagatc aaaatattct ttatgtaggt attgttaaaa ggctagagcc 3120 tacaagttgc tcttccatgc gttggtcagg gggccctgaa aacactggta atattaagag 3180 tctttctcag ggtaacttaa tgttttctta atgaacagtg tttccagcta caaattcttc 3240 caataaattg tcttcctttt tgaaaagtac tctcatagaa gaaatttagc aatttctcgt 3300 tgactgactc agtctatttt aagtattcag aaaagatttt gatccccatt gagttaatgc 3360 tctgccttga aaattatttt tctgatcctt gttagtgata acattttttt tctactgaag 3420 gtcagaggat aggaaacaag tatttctctt ctggtataca tgtaatgtat tctgtaaaaa 3480 agtattcata ttggcaattt tagttaggca taatattgtg gttgtaattt ttaaaactta 3540 gtgttttgtc tgattaaagc aggcactgat cagggtatct cctaagaggt aattcacttc 3600 ttattccttt ccaataatta ttacattcta aattttcatc tatgagaaat aacaaacaag 3660 aagggaatag aattaaattg gggtataatc taatcttcat tgtttaaatg gtttgccttc 3720 tcaccattga agccattttt ttatagcctc agaaagagga aataatgcct ccaccatttt 3780 ctacctggtg acttgaaaat tgaactttta agttaggaag aagttagagt cagggaactt 3840 gtataccact atctatgcag cattgttata gtctgattat ttctgtgttt tgaatatgat 3900 tttcctaatg ctctaaataa aatttttc 3928 <210> 25 <211> 1542 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3344986CB1 <400> 25 atgtaccacc gcgtccgcat gtcatgagtg gaggagtcct tctccctgtc aaccccagac 60 catcccccaa cacctccctg aaattcctgc aaggtcaggc cgatctcagg ctctgactcc 120 tttcactagc ctttctggtg atgtgatctt acccagcttc ctgtttgtct tcctgagaga 180 cagtagattt agaaagtgag gatcagaggg tggaaaataa aagctgtggt ccccaggagt 240 cctgaacatc tgggggcagc gggaaaacat gagtgactcc aaggaaccaa gggtgcagca 300 gctgggcctc ctggggtgtc ttggccatgg cgccctggtg ctgcaactcc tctccttcat 360 gctcttggct ggggtcctgg tggccatcct tgtccaagtg tccaaggtcc ccagctccct 420 aagtcaggaa caatccgagc aagacgcaat ctaccagaac ctgacccagc ttaaagctgc 480 agtgggtgag ctctcagaga aatccaagct gcaggagatc taccaggagc tgacccagct 540 gaaggctgca gtgggtgagt tgccagagaa atccaagctg caggagatct accaggagct 600 gacccggctg aaggctgcag tgggtgagtt gccagagaaa tccaagctgc aggagatcta 660 ccaggagctg acccggctga aggctgcagt gggtgagttg ccagagaaat ccaagctgca 720 ggagatctac caggagctga cccggctgaa ggctgcagtg ggtgagttgc cagaccagtc 780 caagcagcag caaatctacc aggagctgac cgatttgaag actgcatttg aacgcctgtg 840 ccgccactgt cccaaggact ggacattctt ccaaggaaac tgttacttca tgtctaactc 900 ccagcggaac tggcacgact ccgtcaccgc ctgccaggaa gtgagggccc agctcgtcgt 960 aatcaaaact gctgaggagc agaacttcct acagctgcag acttccagga gtaaccgctt 1020 ctcctggatg ggactttcag acctaaatca ggaaggcacg tggcaatggg tggacggctc 1080 acctctgtca cccagcttcc agcggtactg gaacagtgga gaacccaaca atagcgggaa 1140 77/7 j tgaagactgt gcggaattta gtggcagtgg ctggaacgac aatcgatgtg acgttgacaa 1200 ttactggatc tgcaaaaagc ccgcagcctg cttcagagac gaatagttgt ttccctgcta 1260 gcctcagcct ccattgtggt atagcagaac ttcacccact tctacacccc gtgcaccctt 1320 ttgactgggg acttgctggt tgaaggagct catcttgcag gctggaagca ccagggaatt 1380 aattccccca gtcaaccaat ggcatccaga gagggcatgg aggctccata caacctcttc 1440 cacccccaca tctttctttg tcctatacat gtcttccatt tggctgtttc tgagttgtag 1500 cctttataat aaagtggtaa atgttgtaac tgcaaaaaaa as 1542 <210> 26 <211> 1264 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3576503081 <400> 26 cggacttttt ctgtggcgca gcttctccgc ccgagccgcg cgcggagctg ccgggggctc 60 cttagcaccc gggcgccggg gccctcgccc ttccgcagcc ttcactccag ccctctgctc 120 ccgcacgcca tgaagtcgcc gttctaccgc tgccagaaca ccacctctgt ggaaaaaggc 180 aactcggcgg tgatgggcgg ggtgctcttc agcaccggcc tcctgggcaa cctgctggcc 240 ctggggctgc tggcgcgctc ggggctgggg tggtgctcgc ggcgtccact gcgcccgctg 300 ccctcggtct tctacatgct ggtgtgtggc ctgacggtca ccgacttgct gggcaagtgc 360 ctcctaagcc cggtggtgct ggctgcctac gctcagaacc ggagtctgcg ggtgcttgcg 420 cccgcattgg acaactcgtt gtgccaagcc ttcgccttct tcatgtcctt ctttgggctc 480 tcctcgacac tgcaactcct ggccatggca ctggagtgct ggctctccct agggcaccct 540 ttcttctacc gacggcacat caccctgcgc ctgggcgcac tggtggcccc ggtggtgagc 600 gccttctccc tggctttctg cgcgctacct ttcatgggct tcgggaagtt cgtgcagtac 660 tgccccggca cctggtgctt tatccagatg gtccacgagg agggctcgct gtcggtgctg 720 gggtactctg tgctctactc cagcctcatg gcgctgctgg tcctcgccac cgtgctgtgc 780 aacctcggcg ccatgcgcaa cctctatgcg atgcaccggc ggctgcagcg gcacccgcgc 840 tcctgcacca gggactgtgc cgagccgcgc gcggacggga gggaagcgtc ccctcagccc 900 ctggaggagc tggatcacct cctgctgctg gcgctgatga ccgtgctctt cactatgtgt 960 tctctgcccg taatttatcg cgcttactat ggagcattta aggatgtcaa ggagaaaaac 1020 aggacctctg aagaaccaga gagacctccg agccttgcga tttctatctg tgatgtcaat 1080 tgtggaccct tggattctta tcattttcag atctccagta tttcggatat tttttcacaa 1140 gatttttcat tagacctctt aggtacagga gccgatgcag caattccact aacatggaat 1200 ccagtctgtg acagtgtttt tcactctgtg gtaagctgag gagtgtctga catgcggtgg 1260 tggt

Claims (20)

What is claimed is:

1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ
ID NO:12, SEQ ID NO:13, and fragments thereof.

2. A substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of claim 1.

3. An isolated and purified polynucleotide encoding the polypeptide of claim 1.

4. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 3.

5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3.

6. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 3.

7. A method for detecting a polynucleotide. the method comprising the steps of:
(a) hybridizing the polynucleotide of claim 6 to at least one nucleic acid in a sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of the polynucleotide in the sample.

8. The method of claim 7 further comprising amplifying the polynucleotide prior to hybridization.

9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26 and fragments thereof.

10. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 9.

11. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 9.

12. An expression vector comprising at least a fragment of the polynucleotide of claim 3.

13. A host cell comprising the expression vector of claim 12.

14. A method for producing a polypeptide, the method comprising the steps of:
a) culturing the host cell of claim 13 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.

15. A pharmaceutical composition comprising the polypeptide of claim 1 in conjunction with a suitable pharmaceutical carrier.

16. A purified antibody which specifically binds to the polypeptide of claim 1.

17. A purified agonist of the polypeptide of claim 1.

18. A purified antagonist of the polypeptide of claim 1.

19. A method for treating or preventing a disorder associated with decreased expression or activity of HCSRP, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 15.

20. A method for treating or preventing a disorder associated with increased expression or activity of HCSRP, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 18.